TO TREAT CANCER

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Patent Application Serial No. 63/313,105, filed on February 23, 2022. The disclosure of the prior application is considered part of (and is incorporated by reference in) the disclosure of this application.

TECHNICAL FIELD

This document relates to methods and materials for treating cancer (e.g., Wnt-activated and Cetuximab-resistant KRAS mutant cancers such as colorectal adenocarcinomas). For example, this document provides methods and materials for using one or more DDR1 inhibitors and one or more EGFR inhibitors to treat cancers that exhibit little or no response to treatment with an EGFR inhibitor alone.

BACKGROUND INFORMATION

Colorectal cancer (CRC) is the third most common cancer worldwide, and the second highest in cancer mortality after lung cancer. When diagnosed early, the 5 -year survival rate is 91% for localized cancer and 70% for cancer with loco-regional invasion. However, it declines to 15% in advanced or metastatic cases, which includes 25% of patients at the point of diagnosis (Keum et al., Nat Rev Gastroenterol Hepatol., 16(12):713-732 (2019)). Hypermutation status in colorectal adenocarcinoma (COAD) tumors include changes in the WNT, MAPK, PI3K, TGF-P and p53 pathways. Mutations in WNT pathway regulators, such as loss-of-function mutations in Adenomatous polyposis coli (APC) gene and activating mutations in WNT effector gene CTNNB1 (encoding beta-catenin) are the prime trigger for tumorigenesis, and, APC mutations being subtype independent, play a central role in colon cancer development. Activating mutations in Kirsten rat sarcoma virus oncogene (KRAS), and its downstream effectors like, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit-a (PIK3CA), and Serine/Threonine- Kinase B-Raf (BRAF) serve as subtype specific oncogenic drivers, and KRAS mutations account for almost 40% of observed genetic alterations in CRC (Kuipers et al., Nat Rev Dis Primers, 1 : 15065 (2015); Xue et al, Curr Probl Surg., 55(3):76-l 16 (2018); Sztupinszki et al., SciRep., 6:37169 (2016); and Buikhuisen et al., Oncogenesis, 9:66 (2020)). The primary management of colorectal cancer includes surgery, neo-adjuvant chemo-radiation therapy, and administration of an anti-EGFR antibody such as Cetuximab. These treatment strategies however continue to evolve due to molecular profiling of new genetic alterations and emergence of acquired resistance and tumor progression to metastasis that stays refractory to the current standard of care (Buikhuisen et al., Oncogenesis, 9:66 (2020); Ciombor and Bekaii-Saab, Oncologist, 23(l):25-34 (2018); Modest and Pant, Eur J Cancer, 109:70-83 (2019); and Lo Nigro et al., World J Gastroenterol., 22(30):6944-6954 (2016)).

SUMMARY

This document provides methods and materials for using synergistic drug combinations to treat cancer within a mammal (e.g., a human). For example, this document provides methods and materials for using one or more (e.g., one, two, three, four, or more) DDR1 inhibitors and one or more (e.g., one, two, three, four, or more) EGFR inhibitors to treat cancers that exhibit little or no response to treatment with an EGFR inhibitor alone (e.g., a Wnt-activated and Cetuximab-resistant KRAS mutant colorectal adenocarcinoma).

As described herein, one or more (e.g., one, two, three, four, or more) DDR1 inhibitors and one or more (e.g., one, two, three, four, or more) EGFR inhibitors can be used in combination to treat cancer (e.g., reduce the number of cancer cells within a mammal) in a synergistic manner that outperforms the use of either inhibitor type alone.

In general, one aspect of this document features methods for treating cancer in a mammal where cancer cells of the cancer have a KRAS mutation and resistance to treatment with an EGFR inhibitor alone. The methods can include, or consist essentially of, administering, to a mammal having cancer where cancer cells of the cancer have a KRAS mutation and resistance to treatment with an EGFR inhibitor alone, one or more DDR1 inhibitors and one or more EGFR inhibitors, where the number of cancer cells within the mammal is reduced. The method can include identifying the cancer cells as having the KRAS mutation. The method can include determining that the cancer cells are resistant to treatment with the EGFR inhibitor alone. The one or more EGFR inhibitors can includes Lapatinib, Afatinib, and/or Sapitinib. The method can include administering Lapatinib. The method can include administering Afatinib. The one or more DDR1 inhibitors can include Bafetinib, Ponatinib, DDR1 7rh, VU6015929, Nilotinib, Sorafenib, PLX8394, and/or Dactolisib. The one or more DDR1 inhibitors can include PLX8394, Bafetinib, Ponatinib, VU6015929, DDR1 7rh, and/or Nilotinib. The one or more DDR1 inhibitors can include Bafetinib, Ponatinib, DDR1 7rh, VU6015929, and/or Dactolisib. The ratio of the one or more EGFR inhibitors administered to the mammal to the one or more DDR1 inhibitors administered to the mammal can be about 5: 1, about 4: 1, about 3:1, about 2:1, about 1 : 1, or about 1 :2. The Lapatinib and PLX8394 can be administered to the mammal. The ratio of Lapatinib administered to the mammal to PLX8394 administered to the mammal can be about 2: 1. The Lapatinib and Bafetinib can be administered to the mammal. The ratio of Lapatinib administered to the mammal to Bafetinib administered to the mammal can be about 1 :1 or about 1 :2. The Lapatinib and Ponatinib can be administered to the mammal. The ratio of Lapatinib administered to the mammal to Ponatinib administered to the mammal can be about 2:1 or about 3: 1. The Lapatinib and DDR1 7rh can be administered to the mammal. The ratio of Lapatinib administered to the mammal to DDR1 7rh administered to the mammal can be about 2: 1. The Lapatinib and Nilotinib can be administered to the mammal. The ratio of Lapatinib administered to the mammal to Nilotinib administered to the mammal can be about 1 : 1. The method can include administering a Src inhibitor to the mammal. The Src inhibitor can be KB SRC-4. The method can include administering a degrader of BCR-ABL to the mammal. The degrader of BCR-ABL can be GMB475. The cancer can be a primary tumor. The cancer can be a metastasis. The cancer can be colorectal cancer, glioblastoma, lung cancer, pancreatic cancer, or breast cancer. The cancer can be a colorectal cancer. The cancer can be a glioblastoma. The KRAS mutation can be a KRAS G13D mutation, a KRAS G12V mutation, a KRAS G12A mutation, a KRAS G12C mutation, a KRAS G12D mutation, a KRAS G12R mutation, a KRAS Q61L mutation, a KRAS Q61K mutation, a KRAS Q61R mutation, a KRAS Q61 V mutation, a KRAS Q61 A mutation, or a KRAS Q61C mutation. The cancer can be a Cetuximab-resistant cancer. The cancer can be a chemo-radio-resistant cancer. The mammal can have been previously treated with a cancer treatment for the cancer. The mammal can have been previously treated with an EGFR inhibitor treatment. The mammal can have been previously treated with chemotherapy. The method can include administering the one or more DDR1 inhibitors at least once a week for six months to three years. The method can include administering the one or more EGFR inhibitors at least once a week for six months to five years. The administering of the one or more DDR1 inhibitors can be via intravenous, subcutaneous, intraperitoneal, rectal, or oral administration. The administering of the one or more EGFR inhibitors can be via intravenous, subcutaneous, intraperitoneal, rectal, or oral administration. The one or more EGFR inhibitors and the one or more DDR1 inhibitors can be administered simultaneously to the mammal. The one or more EGFR inhibitors can be administered to the mammal prior to the one or more DDR1 inhibitors. The one or more EGFR inhibitors can be administered to the mammal after the one or more DDR1 inhibitors. The administration of the one or more EGFR inhibitors and the one or more DDR1 inhibitors can have a synergistic effect within the mammal. The administering can reduce the volume of the cancer within the mammal. The mammal can be a human. The one or more DDR1 inhibitors and the one or more EGFR inhibitors can be administered to the mammal as the sole active ingredients against the cancer.

In another aspect, this document features a DDR1 inhibitor for use in increasing susceptibility of a cancer within a mammal to an EGFR inhibitor. The cancer can be resistant to exposure to the EGFR inhibitor alone.

In another aspect, this document features a DDR1 inhibitor for use in combination with an EGFR inhibitor to treat a cancer within a mammal. The cancer can be resistant to exposure to the EGFR inhibitor alone.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and methods are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

DESCRIPTION OF THE DRAWINGS

Figure 1A-1D: Figure 1A. Representative bright-field images ofHCT116 spheroids cultured in media having, nitrogen supplement (N2), plus EGF and FGF2 (N2EF), taken at time points indicated. Spheroid viability was measured in response to Cetuximab (anti-EGFR) treatment on HCT116 multi- spheroids cultured in N2EF media. Figure IB. Effect of irradiation on growth and survival of HCT116 spheroids administered with IR doses indicated. ROS levels, spheroid viability and Caspase 3/7 activity were measured 24 h and 5 days post IR (experimental scheme in Figure 9C). Significance in change of spheroid viability between 24 h and 5 day post IR dose treatment is indicated (****, p-value < 0.0001). Figure 1C. Drug screening: Inhibitors listed were administered as single agent on HCT116 (N2EF) multi-spheroids, and spheroid viability was estimated. Table includes percent inhibition (%Inh.) observed for each drug when administered at concentrations 25pM, lOpM, and IpM (dose response curves are included in Figure 11). IC50 was not reached for drugs marked with a cross and were excluded further from the study. Figure ID. Single agent spheroid viability curves for WNT pathway inhibitors and, small molecule inhibitors to EGFRZERBB proteins on HCT116 multi-spheroids.

Figure 2A-2C: Figure 2 A Efficacy of the drugs in combination with EGFR small molecule inhibitors (EGFRi). Drugs were administered on multi- spheroid ofHCT116 (N2EF), and spheroid viability was measured. Table indicates percent inhibition obtained for each of the drug alone, or in combination with Lapatinib, Afatinib or Sapitinib. Color-scale of percent inhibition indicates overall combinatorial efficacy of Lapatinib > Afatinib > Sapitinib. In columns to the right, averaged percent inhibition for each drug when administered in combination with all three EGFRi and, fold change between drug+EGFRi vs. drug alone is mentioned. Inhibitors marked with black asterisk are prime 6 combinatorial drugs identified, which showed >70% inhibition with at least two out of three EGFR inhibitors tested. Top-5 inhibitors (marked with f) had averaged percent inhibition > 50, and fold change > 2. Figure 2B. Bar-graphs indicate relative spheroid viability of HCT116 multi-spheroids when administered with the prime 6 combinatorial leads identified. Figure 2C Representative images of HCT116 multi-spheroids obtain by culturing in additional media supplemented with N2, and N21max. Top-5 combinatorial leads identified were validated in combination with EGFR inhibitors (Lapatinib, Afatinib), on HCT116 multi-spheroids cultured in N2N21max. Bar-graphs for combination treatments tested are represented to the right.

Figure 3A-3D. Figure 3A. Representative bright-field images of multi-spheroids obtained for DLD1 cultured in N2N21max media, at times points indicated. Dose response curves (right) indicate the efficacy of WNT pathway inhibitors, and EGFR small molecule inhibitors (Lapatinib, and Afatinib) as single agents on DLD1 spheroid viability. Figure 3B. Combinatorial efficacy of the prime leads identified, when administered on multi-spheroids from DLD1. Bargraphs indicate relative spheroid viability of DLD1, when treated with respective drugs ± EGFR inhibitors (Lapatinib and Afatinib). Table on the right shows percent inhibition values calculated for all the 6 drugs alone, or in combination with EGFRi, Lapatinib and Afatinib. Color-scale indicates Lapatinib to have higher combinatorial efficacy than Afatinib. Figure 3C. Combinatorial efficacy of lapatinib with prime 6 leads was validated in additional line, SW480, harboring KRAS G12V mutation. Representative bright-field images (left) show multi-spheroids for SW480 cultured in N2N21max media at indicated time points. Table enlists KRAS mutations known for the respective cell lines: SW480, having G12V, as opposed to G13D in HCT116 and DLD1. Bar-graphs (right) indicate relative spheroid viability of SW480 when administered with respective drugs ± EGFR inhibitor, Lapatinib. Figure 3D. Table includes averaged percent inhibition obtained for each of the COAD cell line (HCT116, DLD1, SW480) when administered with drugs ± EGFR inhibitor, Lapatinib and Afatinib. Averaged percent inhibition for all three cell lines, and fold-change observed between drug administered in combination (+ EGFRi) vs. drug alone, is mentioned in columns to the right. Inhibitors marked (with symbol, f ) are the top- 5 combinatorial inhibitors identified.

Figure 4A-4L. Figure 4A. Synergy plot for EGFR inhibitor, Lapatinib ± Bafetinib administered to HCT116 multi-spheroids cultured in N2EF media. 3D-surface plot having a rise of the curve in positive XY-axis indicating synergy. Figure 4B. Synergy plot for EGFR inhibitor, Lapatinib ± Ponatinib administered to HCT116 multi-spheroids cultured in N2EF media. 3D- surface plot having a rise of the curve in positive XY-axis indicating synergy. Figure 4C. Synergy plot for EGFR inhibitor, Lapatinib ± DDR1 7RH administered to HCT116 multispheroids cultured in N2EF media. 3D-surface plot having a rise of the curve in positive XY- axis indicating synergy. Figure 4D. Synergy plot for EGFR inhibitor, Lapatinib ± VU6015929 administered to HCT116 multi-spheroids cultured in N2EF media. 3D-surface plot having a rise of the curve in positive XY-axis indicating synergy. Figure 4E. Synergy plot for EGFR inhibitor, Lapatinib ± PLX894 administered to HCT116 multi-spheroids cultured in N2EF media. 3D- surface plot having a rise of the curve in positive XY-axis indicating synergy. Figure 4F. Synergy plot for EGFR inhibitor, Afatinib ± Bafetinib administered to HCT116 multi-spheroids cultured in N2EF media. 3D-surface plot having a rise of the curve in positive XY-axis indicating synergy. Figure 4G. Synergy plot for EGFR inhibitor, Afatinib ± Ponatinib administered to HCT116 multi-spheroids cultured in N2EF media. 3D-surface plot having a rise of the curve in positive XY-axis indicating synergy. Figure 4H. Synergy plot for EGFR inhibitor, Afatinib ± DDR1 7RH administered to HCT116 multi-spheroids cultured in N2EF media. 3D- surface plot having a rise of the curve in positive XY-axis indicating synergy. Figure 41. Synergy plot for EGFR inhibitor, Afatinib ± VU6015929 administered to HCT116 multi-spheroids cultured in N2EF media. 3D-surface plot having a rise of the curve in positive XY-axis indicating synergy. Figure 4 J. Synergy plot for EGFR inhibitor, Afatinib ± PLX8394 administered to HCT116 multi-spheroids cultured in N2EF media. 3D-surface plot having a rise of the curve in positive XY-axis indicating synergy. Figure 4K. Tabulation of the cumulative synergy score for the teste drug combinations. A matrix for synergy score values and a matrix for percent inhibition obtained for all 25 combination treatments, has been created for each of the synergy experiment performed, and included in Figure 13. Figure 4L. Analysis of targets for top-5 combinatorial leads and targets common among them. DDR1, discoidin domain receptor 1 (neurotrophic receptor tyrosine kinase, NTRK4), came-up as the most prevalent target among the combinatorial leads.

Figure 5A-5C. Figure 5a. Genomic associations of gene DDR1 with genes relating to other targets of the combinatorial (top-5) leads identified. Correlation matrix represents correlation coefficients, R-Pearson (Rp) or R-Spearman, (Rs), obtained between the mRNA expression of each of the genes indicated on y-axis and the gene DDR1. Datasets evaluated for tumors vs. normal, include colorectal adenocarcinoma (COAD), Glioblastoma (GBM), Colon (normal) and Brian cortex (normal) utilizing GEPIA; and Pan-Cancer Atlas using cBioPortal. All correlation coefficient values included are Rp, except for pan-cancer atlas, for which both Rp and Rs values were considered, due to intrinsic variability and heterogeneity among various tumor types. The correlation table shows, DDR1 to positively correlate with EGFR, BCR and, ABL1, and downstream mediators of KRAS signaling (BRAF, PIK3CA, MT OR) in both tumors (COAD and GBM), and, DDR1 correlated with genes EGFR, ERBB2, ERBB4, BRAF, BCR, and genes of sternness, polarity and adhesion (SOX9, VANGL2, CDH1) in pan-cancer. Figure 5B. DDR1/BCR-ABL signaling networks using STRING database: The network (32 nodes and 112 edges), includes proteins reported to be engaging in physical interactions with high confidence (score > 0.7). Clustering performed utilizing MCL method revealed eight clusters listed at the top of the protein network. The clustering indicates SHC1 to be the adaptor protein that links DDR1 to other signaling pathways, including EGFR/ERBB2 which activate KRAS downstream; PTPN11 (activator of KRAS signaling) is identified as another DDR1 interaction partner. Interaction sources utilized in STRING database involved: Textmining, Experiments, Co-expression, Databases, Neighborhood, Co-occurrence (but, excluding gene-fusions). Figure 5C. Based on Pathway Commons database, the signaling relay from DDR1 to BCR and KRAS is predicted to engage adaptor proteins SHC1, GRB2 and SOS1. The protein-interactome of DDR1, BCR and ABL1 with selective proteins on the right show: physical interactions of DDR1 with ERBB2 and SHC1; physical and functional interactions of BCR with EGFR, ERBB2, adaptors (SHC1, GRB2, SOS1), KRAS, and ABL1; physical interactions of ABL1 with EGFR, ERBB2, adaptors (SHC1, GRB2, SOS1), and CTNNB1 and function interactions of ABL1 with SOS1, and CTNNB1. Together this indicates signaling relay from DDR1 (as homodimer or heterodimer with EGFR/ERBB2) via SHC1, GRB2, SOS1 to BCR and, from BCR-ABL1- CTNNB1 complex to KRAS.

Figure 6A-6E. Figure 6A Combinatorial efficacy of DDR1/BCR-ABL1 multi-tyrosine kinase inhibitors (Dasatinib, Imatinib and Nilotinib) and compound drugs, GMB475 and KB- SRC4 (BCR-ABL1 specific PROTAC, and SRC inhibitor respectively), when administered with EGFRi (lapatinib, Afatinib) on multi- spheroids ofHCT116. Representative images (left), and Bar-graphs (right) indicate spheroid viability, with percent inhibition obtained enlisted in table at the bottom. Bar-graphs showing relative caspase 3/7 activity (normalized to viability) in multispheroids at the time-point of day2 post drug treatment are included in Figure 18 A. Figure 6B. Clonal cell proliferation assay performed for HCT116 cells for drugs, Bafetinib and Nilotinib, in combination with Lapatinib and clonal growth measured as percent confluence, using incucyte. Representative images (right) for clonal cell proliferation and growth obtained at time points dayO, day3 and day5, when administered with drugs as single agents or in combination, pseudocolored utilizing the incucyte’ s inbuilt Clonal-dilution module. Figure 6C. Efficacy of Nilotinib in combination with Lapatinib validated on viability of multi-spheroids obtained from KRAS- mutant line, SW480 (KRASG12V). Figure 6D. Efficacy of DDR1 specific inhibitor (DDR1 7RH) in combination with Lapatinib on viability of multi- spheroids obtained from additional tumor model of GBM, utilizing U251 cells. Figure 6E. Representative bright-field images of U251 (GBM) spheroids treated with drugs Lapatinib and Nilotinib, as single agent or in combination, at day5 post-treatment. Graph (middle) shows reduction in spheroid total area over 5 days post-treatment. Bar-graph (right) indicates loss of spheroid cell viability measured on day5.

Figure 7A-7D. Figure 7A. Targeting DDR1/BCR-ABL with EGFR/ERBB2 against radioresistance: HCT116 multi-spheroids that recovered from radiation stress induced by administering IR-doses from OGy up to 20Gy as indicated were measured for, Spheroid viability and Caspase 3/7 activity, at Day5 post-IR. P-values for combination treatment of drugs (Bafetinib or Nilotinib) with Lapatinib are marked as asterisk, at each of the respective IR-doses administered. Figure 7B. Combinatorial efficacy of brain penetrable DDR1/BCR-ABL inhibitor Nilotinib plus EGFR-inhibitor Lapatinib on spheroid formation of patient-derived GBM (GBM- PD) lines, GBM965 and QNS108. Figure 7C. Combinatorial efficacy of brain penetrable DDR1/BCR-ABL inhibitor Nilotinib plus EGFR-inhibitor Lapatinib on viability of spheroids of patient-derived GBM (GBM-PD) lines, GBM965 and QNS108. Figure 7D. Proposed model illustrating the signaling networks involving DDR1, SRC kinases, BCR-ABL-Pcatenin, EGFR/ERBB, and PI3K/AKT/mT0R with KRAS proteins, KRASWT and KRASmut and, cite of action of the leads identified. Illustration (left) shows KRASWT cross-talks: Activated DDR1 relays its effect via adaptor proteins SHC1/GRB2/SOS1 causing dissociation of BCR-ABL- Pcatenin complex (inactive) into activated BCR-ABL proteins and freed P-catenin, which is now available to translocate to nucleus to cause transcription of WNT -target genes. BCR and ABL proteins interact to cause activation of KRASWT and PIK3/AKT signaling axis. SRC kinases activated downstream of DDR1 can engage with KRAS in a positive feed-back loop. Additionally, EGFR/ERBB2 receptor complex can activate KRAS-dependent MAPK signaling, and PIK3/AKT/mT0R signaling; and, activated KRAS can also activate PI3K/AKT/mT0R axis. Blue and Green arrows indicate cause-effect interactions, with Green-arrows indicating input signals that activate KRASWT and Blue-arrows indicating output signals from KRAS to other signaling intermediates. Illustration (right) depicts these signaling interactions with KRASmut protein which is nearly constitutively active, thus, no input signals (green-arrows) required. Targeting the network of hyperactivated KRAS, or KRASmut comprises of the scheme: (1) combinatorial targeting of DDR1 & BCR-ABL1 by specific inhibitors of DDR1, or DDR1/BCR- ABL by multi-tyrosine kinase inhibitors (Bafetinib, Ponatinib, Nilotinib), in combination with EGFR inhibitors, Lapatinib/ Afatinib. Other potential combinations identified are represented as schemes (2) and (3): involving PI3K/mTOR inhibition by Dactolisib, in combination with EGFR inhibitors, Lapatinib/Afatinib or KRAS/RAF axis inhibition by PLX8394 (which would inhibit MAPK signaling downstream of KRAS) in combination with EGFR inhibitors, Lapatinib/Afatinib. These combinatorial schemes can be harnessed to overcome the treatment resistance observed in WNT-activated (stem-like) KRAS mutant tumors.

Figure 8. Radio-sensitivity curves for GBM cell lines (QNS695 and QNS108) following a single dose of 0 Gy, 2 Gy, 4 Gy, or 6 Gy (275.1 cGy/min) with an X-rad® 1600 (Precision X- Ray).

Figure 9A-9C. Figure 9A. Illustration to represent that, mutant (inactive) APC protein and high-nuclear P-catenin co-operate bidirectionally with upregulated KRAS signaling. Together, WNT effector activation, and activated KRAS protein mediate radioresistance and cetuximab resistance observed in advanced refractory tumors. To the right is study aim stated. Figure 9B. Illustration to depict the study work-flow. Figure 9C. Experimental scheme to investigate internal resistance to radiation in HCT116 multi-spheroids and scheme utilized to perform drug treatments and multi- spheroid viability assays in HCT116.

Figure 10A-10E. Figure 10A. Representative bright field images (4x) showing spheroids obtained from floating cultures of HCT116 and DLD1 in media conditions indicated. Figure 10B. Three independent Bright field images (4x, and lOx) of multi- spheroids obtained from HCT116 and DLD1 under indicated media conditions, at day3 in culture. Figure 10C. Bright field (BF) images at 4x, and 40x, for HCT116 cultured in N2EF vs. N2N21max media, and DLD1 in N2N21max media. Approximately 15 independent images under each condition were taken using EVOS FL microscope, and the spheroid diameter was averaged. Figure 10D. Comparing the Spheroid growth parameters, spheroid Average Area (pm ² ) and, spheroid total area (pm ² /image) and spheroid average eccentricity for HCT116 and DLD1 multi- spheroids cultured in 96 well plate, under media conditions indicated, and over a period of 168 hours (day 7). Figure 10E. Graph (left): Increase in Spheroid average area for SW480 cell line over a period of 5 days. Graph (right), Spheroid average eccentricity for SW480.

Figure 11A-11E. Figure 11 A. Spheroid viability curves for colorectal adenocarcinoma lines HCT116 (cultured in media N2EF) when administered with drugs as single agents. Figure 1 IB. Spheroid viability curves for colorectal adenocarcinoma lines HCT116 (cultured in media N2N21max) when administered with drugs as single agents. Figure 11C. Spheroid viability curves for colorectal adenocarcinoma lines DLD1 when administered with drugs as single agents. Figure 1 ID. Spheroid viability curves for colorectal adenocarcinoma lines SW480 when administered with drugs as single agents. Figure 1 IE. Spheroid viability curves for glioblastoma lines (GBM cell line: U251, and, Patient-derived GBM lines: GBM965 and QNS108) when administered with respective drugs as single agent.

Figure 12A-12B. Figure 12A. Bar-graphs showing spheroid viability for HCT116 (cultured in media N2EF) when administered with drugs as single agents, or in combinations at concentrations < IC50. Doses at which the drugs were combined, and percent inhibition estimated has been included. Figure 12B. Spheroid viability in response to Cetuximab (anti- EGFR) treatment on HCT116 multi- spheroids cultured in N2N21max media (left). To the right, is effect of irradiation on growth and survival of HCT116 spheroids (cultured in N2N21max). ROS levels, spheroid viability and Caspase 3/7 activity were measured 24 hours post-IR, and, at Day 5 post-IR (experimental scheme in Figure 9C). Significance in change of spheroid viability between 24 hour-post IR and Day 5 post-IR at respective IR-dose treatment is indicated (****, p- value < 0.0001).

Figure 13A-13J. Figure 13 A. Matrix of synergy score values for the combination of Lapatinib and Bafetinib at 25 combination treatments. Figure 13B. Matrix of synergy score values for the combination of Lapatinib and Ponatinib at 25 combination treatments. Figure 13C. Matrix of synergy score values for the combination of Lapatinib and DDR1 7RH at 25 combination treatments. Figure 13D. Matrix of synergy score values for the combination of Lapatinib and VU6015929 at 25 combination treatments. Figure 13E. Matrix of synergy score values for the combination of Lapatinib and PLX8394 at 25 combination treatments. Figure 13F. Matrix of synergy score values for the combination of Afatainib and Bafetinib at 25 combination treatments. Figure 13G. Matrix of synergy score values for the combination of Afatinib and Ponatinib at 25 combination treatments. Figure 13H. Matrix of synergy score values for the combination of Afatinib and DDR1 7RH at 25 combination treatments. Figure 131. Matrix of synergy score values for the combination of Afatinib and VU6015929 at 25 combination treatments. Figure 13 J. Matrix of synergy score values for the combination of Afatinib and PLX8394 at 25 combination treatments.

Figure 14A-14J. Figure 14A. Matrix of percent inhibition values for the combination of Lapatinib and Bafetinib at 25 combination treatments. Figure 14B. Matrix of percent inhibition values for the combination of Lapatinib and Ponatinib at 25 combination treatments. Figure 14C. Matrix of percent inhibition values for the combination of Lapatinib and DDR1 7RH at 25 combination treatments. Figure 14D. Matrix of percent inhibition values for the combination of Lapatinib and VU6015929 at 25 combination treatments. Figure 14E. Matrix of percent inhibition values for the combination of Lapatinib and PLX8394 at 25 combination treatments. 14F. Matrix of percent inhibition values for the combination of Afatainib and Bafetinib at 25 combination treatments. Figure 14G. Matrix of percent inhibition values for the combination of Afatinib and Ponatinib at 25 combination treatments. Figure 14H. Matrix of percent inhibition values for the combination of Afatinib and DDR1 7RH at 25 combination treatments. Figure 141. Matrix of percent inhibition values for the combination of Afatinib and VU6015929 at 25 combination treatments. Figure 14J. Matrix of percent inhibition values for the combination of Afatinib and PLX8394 at 25 combination treatments.

Figure 15A-15J. Figure 15 A. Dot plots showing relative spheroid viability obtained for all 5 doses of Lapatinib and Bafetinib (administered as single agent), and for all 25 combination treatments done (from Figures 13 and 14) and bar- graphs, showing the relative spheroid viability in the presence of 7 pM Lapatinib and/or 6.5 pM Bafetinib. Figure 15B. Dot plots showing relative spheroid viability obtained for all 5 doses of Lapatinib and Ponatinib (administered as single agent), and for all 25 combination treatments done (from Figures 13 and 14) and bargraphs, showing the relative spheroid viability in the presence of 7 pM Lapatinib and/or 3 pM Ponatinib. Figure 15C. Dot plots showing relative spheroid viability obtained for all 5 doses of Lapatinib and DDR1 7RH (administered as single agent), and for all 25 combination treatments done (from Figures 13 and 14) and bar-graphs, showing the relative spheroid viability in the presence of 2.4 pM Lapatinib and/or 3 pM DDR1 7RH. Figure 15D. Dot plots showing relative spheroid viability obtained for all 5 doses of Lapatinib and VU6015929 (administered as single agent), and for all 25 combination treatments done (from Figures 13 and 14) and bar-graphs, showing the relative spheroid viability in the presence of 10 pM Lapatinib and/or 6 pM VU6015929. Figure 15E. Dot plots showing relative spheroid viability obtained for all 5 doses of Lapatinib and PLX8394 (administered as single agent), and for all 25 combination treatments done (from Figures 13 and 14) and bar-graphs, showing the relative spheroid viability in the presence of 15 pM Lapatinib and/or 6 pM PLX8394. Figure 15F. Dot plots showing relative spheroid viability obtained for all 5 doses of Afatinib and Bafetinib (administered as single agent), and for all 25 combination treatments done (from Figures 13 and 14) and Bar-graphs, showing the relative spheroid viability in the presence of 4.8 pM Afatinib and/or 13 pM Bafetinib. Figure 15G. Dot plots showing relative spheroid viability obtained for all 5 doses of Afatinib and Ponatinib (administered as single agent), and for all 25 combination treatments done (from Figures 13 and 14) and bar-graphs, showing the relative spheroid viability in the presence of 2.4 pM Afatinib and/or 3 pM Ponatinib. Figure 15H. Dot plots showing relative spheroid viability obtained for all 5 doses of Lapatinib and DDR1 7RH (administered as single agent), and for all 25 combination treatments done (from Figures 13 and 14) and bar-graphs, showing the relative spheroid viability in the presence of 2.4 pM Afatinib and/or 3 pM DDR1 7RH. Figure 151. Dot plots showing relative spheroid viability obtained for all 5 doses of Afatinib and VU6015929 (administered as single agent), and for all 25 combination treatments done (from Figures 13 and 14) and bar-graphs, showing the relative spheroid viability in the presence of 10 pM Afatinib and/or 6 pM Bafetinib. Figure 15J. Dot plots showing relative spheroid viability obtained for all 5 doses of Afatinib and PLX8394 (administered as single agent), and for all 25 combination treatments done (from Figures 13 and 14) and bar-graphs, showing the relative spheroid viability in the presence of 5 pM Afatinib and/or 6.5 pM PLX8394.

Figure 16A-16H. Figure 16A. Synergy plots for Lapatinib ± Nilotinib, for HCT116 multi-spheroids cultured in N2EF media. Figure 16B. Dot plots showing relative HCT116 spheroid viability obtained for all 5 doses of Lapatinib and Nilotinib (administered as single agent), and for all 25 combination treatments done (from 16C and 16D) and bar-graphs, showing the relative spheroid viability in the presence of 3.75 pM Lapatinib and/or 3 pM Nilotinib. Figure 16C. Cumulative synergy scores obtained for each drug combination in multi- spheroid HCT116 cultured in N2EF media. Figure 16D. Cumulative antagonism scores obtained for each drug combination in multi- spheroid HCT116 cultured in N2EF media. Figure 16E. Synergy plots for Lapatinib ± Nilotinib, for U251 multi-spheroids. Figure 16F. Dot plots showing relative U251 spheroid viability obtained for all 5 doses of Lapatinib and Nilotinib (administered as single agent), and for all 25 combination treatments done (from 16G and 16H) and bar-graphs, showing the relative spheroid viability in the presence of 3.75 pM Lapatinib and/or 3 pM Nilotinib. Figure 16G. Cumulative synergy scores obtained for each drug combination in multispheroid U251. Figure 16H. Cumulative antagonism scores obtained for each drug combination in multi-spheroid HCT116 cultured in N2EF media. . *p < 0.05, **p < 0.01, ***p < 0.001,

****p < 0.0001.

Figure 17A-17D. Figure 17A. Relative gene expression: The Log2 (TPM+1) values for mRNA transcripts for selected genes compared in two biological states, normal and tumor, for both colorectal adenocarcinoma (COAD) and glioblastoma (GBM), obtained utilizing Gene expression profiling interactive analysis, GEPIA, that links the datasets from TCGA (for tumor) and GTEx (for normal). Red arrows indicate genes upregulated in COAD. Figure 17B. Genomic associations of gene KRAS with genes relating to other targets of the combinatorial (top-5) drug- leads identified. Correlation matrix represents correlation coefficients, R-Pearson (Rp) or R- Spearman, (Rs), obtained between the mRNA expression of each of the genes indicated on y-axis and the gene KRAS. Datasets evaluated for tumors vs. normal were obtained utilizing genomics portals: GEPIA; and cBioPortal. KRAS expression correlated positively with ERBB2/3, DDR1, SOX9, and cell adhesion proteins (CTNNB1, CTNNA1, CDH1) in normal colon, and positively associated with DDR1 in COAD and GBM. Overall, KRAS had an association with BRAF, PIK3CA, SRC, BCR, APC, and catenin (CTNNB1, CTNNA1) in both these tumor models. KRAS associated with ABL1, and VANGL2 in COAD and GBM in a tumor specific manner, and, with BRAF, PIK3CA, APC and CTNNB1 in pan-cancer. Figure 17C. Correlation matrix for the correlation coefficients (Pearson, Rp) obtained between the mRNA expression of each of the genes indicated on y-axis, vs. genes indicated on x-axis. Datasets evaluated are colorectal adenocarcinoma (COAD), and Colon (normal) utilizing GEPIA. Correlation coefficient values (Rp) are included at the one-half of the matrix (bottom-half triangle) and their P-values are included on the other-half of the matrix (top-half triangle), as illustrated at right corner, in black. Figure 17D. Putative signaling pathway and its intermediates that emerge to play a role in COAD based on the significant correlation coefficients (Rp values) obtained, (cell membrane receptors on upper half, and intracellular signaling mediators in bottom half). Arrows indicate interconnections, with Rp values stated, and p-values marked as asterisks.

Figure 18A-18D. Figure 18A. Bar-graphs showing relative caspase 3/7 activity (normalized to viability) in multi-spheroids 2 post drug treatment. Figure 18B. Clonal cell proliferation assay performed using Incucyte for HCT116 and CaCo2 treated with drugs selected from Lapatinib, DDR1 7RH, and Lapatinib. Nilotinib had better efficacy, when administered in combination with Lapatinib. DDR1 inhibition by 7RH drug was significantly cytotoxic both as single agent and in combination. P-values are indicated, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Figure 18C. Evaluation of late effects of drug treatment, by having the inhibitors administered on large (over-sized) spheroids. HCT116 spheroids were cultured for 2 days, images were acquired at 48 hours, and then, continued to be grown until 90 hours (-day 4), when drugs were administered. Spheroids were reimaged at 138 hours (~ day 6), and then lysed for evaluation of viability. Bar-graph shows significant reduction in combinations as compared to drugs administered alone. Figure 18D. U251 spheroids were cultured until day 13, drugs were administered, and then, effect of drugs on spheroid viability was estimated at day 15. Bar-graph shows significant reduction in combinations as compared to drugs administered alone (bargraph).

Figure 19A-19B. Figure 19A. Bar-graphs showing normalized read counts (Scale: Log2) for patient derived GBM line, GBM965 (Gene expression dataset used: GSE144610). The transcriptional expression of proteins that positively activate KRAS (EGFR/ERBB2, BCR- ABL1, CTNNB1, PTPN2, PTPN11) were upregulated, while the negative regulator of KRAS (NF1) had lower expression. Additionally, KRAS downstream targets like, BRAF, PIK3CA, MTOR were upregulated, and the negative regulator of PIK3/AKT pathway, the tumor suppressor PTEN had lower expression reads (***p < 0.001, ****p < 0.0001). Figure 19B. Fold difference between reads (counts-normalized) for every gene with respect to the average number of transcriptional reads (average counts-normalized) observed in the entire dataset. The data is presented along with expression values of three house-keeping genes (ACTB, beta-actin; Glyceraldehyde 3-phosphate dehydrogenase, GAPDH; Ubiquitin, UBB). Key: GBM965 (B), GBM965 unsorted bulk cells; GBM965 (M), GBM965 sorted for having migratory potential.

Figure 20 is a pie chart plotting samples studied.

DETAILED DESCRIPTION

Colorectal cancer (CRC) represented primarily as colorectal adenocarcinoma (COAD) is the second most common cause of cancer deaths worldwide. Multimodality treatment includes neoadjuvant chemoradiation (nCRT) with an anti-EGFR antibody (e.g., Cetuximab) and surgery. However, amplified EGFR, mutations in APC, KRAS, RAF, and/or PI3K/mTOR, and positive feed-back between activated KRAS and WNT-effectors together account for acquired treatment resistance in CRC, and tumor progression to metastasis. Challenges in direct targeting of WNT regulators (APC, P-catenin) and KRAS have caused alternative actionable targets to gain recent attention. Utilizing an unbiased drug-screen, combinatorial targeting of the DDR1/BCR-ABL signaling axis with small molecule inhibitors of EGFR/ERBB2/3 signaling was found to be potentially cytotoxic against multi-spheroids obtained from WNT-activated KRAS-mutant COAD lines (HCT116, DLD1, and SW480), independent of their KRAS-mutation type and status. Based on the data-driven approach using available patient datasets (TCGA), a transcriptomic-correlation matrix between genes for DDR1, EGFR, SRC, BCR, and ABL and cancer stem cell reactivation was constructed. A pathway network construction based on STRING database led to the identification of the targetable protein interactome in mutant KRAS- driven tumors engaging its downstream effectors and complementary cascades, including DDR1, SRC, BCR and ABL proteins. The cytotoxic potential of inhibitor combinations in an additional tumor model of glioblastoma, and in radio-resistant spheroids of HCT116 (COAD) and GBM- PDX was validated in vitro. Collectively, these results demonstrate that combinatorial targeting of DDR 1 /BCR- ABL and EGFR/ERBB2/3 signaling is an effective therapy against stem-like and KRAS-driven chemo-radio-resistant tumors of COAD and GBM.

Amplified EGFR signaling leads to pronounced cancer cell proliferation, survival, and metastasis (Sigismund et al., Mol OncoL, 12(l):3-20 (2018)). Cetuximab (Erbitux) is a chimeric IgGl monoclonal antibody that competes with receptor ligands for binding to EGFR, and is used in combination with chemotherapy, either in first or in second line, or alone in refractory CRC disease. However, Cetuximab resistance is observed with KRAS, NRAS, and/or BRAF gene mutations, all of which lead to aberrant activation of mitogen-activated protein kinase (MAPK) signaling, thus limiting its clinical use to wild-type RAS/RAF tumors (Lo Nigro et al., World J Gastroenterol., 22(30):6944-6954 (2016); Sigismund et al., Mol Oncol., 12(l):3-20 (2018); Chang et al., J Hematol Oncol., 2: 18. doi: 10.1186/1756-8722-2-18 (2009); and Therkildsen et aL, Acta OncoL, 53(7):852-864 (2014)). Phase-I/II trials are undergoing to test the efficacy of small molecule inhibitors of EGFR with Cetuximab in combination to obtain a better treatment outcome (Deeken et al. , Cancer, 121(10): 1645-1653).

Cetuximab-resistant CRC tumors have been shown to have mutational hotspots located in the genomic landscapes of receptor tyrosine kinases (EGFR/ERBB2), RAS and WNT pathways (Bray et al., Sci Rep., 9(1): 15365 (2019)). Amplified WNT (APC ^mut , CTNNBl ^mut ) and KRAS are reported to have a bi-directional co-operative association contributing to acquired Cetuximab resi stance (Lee et al. , Exp Mol Med. , 50(l l): l-12 (2018); Hwang et al., Int J Cancer, 146(10)2877-2890 (2020); and Moon etal., J Natl Cancer Inst., 106(2):djt373 (2014)). Moreover, WNT activation and high nuclear P-catenin levels are a predictor of chemo-radio- resistance in various cancers including CRC, and KRAS mutation is associated with radioresistance in advanced colorectal tumors (Emons et al., Cancer Res., 15(11): 1481-1490 (2017); Gomez-Millan et al., BMC Cancer, 14: 192 (2014); Liu et al., Cancer Cell Int., 18: 156 (2018); and Toulany et al., Radiother Oncol., 76(2): 143-150 (2005)). Several WNT inhibitors are in clinical trials, however, intrinsic high- WNT activity in stem cells and in normal colon/rectum makes direct WNT targeting a challenge (Krishnamurthy et al., Cancer Treat Rev., 62:50-60 (2018); Cheng et al., Biomed Pharmacother., 110:473-481 (2019); and Zhan et al., Oncogene, 36: 1461-1473 (2017)). Additionally, limitations in development of KRAS mutation specific inhibitors makes KRAS an as-yet unbeatable oncogene (Timar and Kashofer, Cancer Metastasis Rev., 39(4): 1029-1038 (2020); Grapsa and Syrigos, Expert Rev Anticancer Ther., 20(6):437-440 (2020); and Fan et aL, Eur J MedChem., 226: 113816 (2021)). Signaling pathways documented to be contributors in CRC development (Cerrito and Grassilli, Biomedicines, 9(5):579 (2021); Xie et al., Sig Transduct Target Ther., 5:22 (2020); Medico et al., Nat Commun., 6:7002 (2015); and Berg et al., Mol Cancer, 16(1): 116 (2017)) were screened with an aim to identify drug combinations with EGFR inhibitors (EGFRi) that could offer a therapeutic advantage to overcome acquired radio-resistance and/or Cetuximab-resistance, utilizing colorectal tumor models harboring a stem-like (WNT activated) and KRAS mutation phenotype.

Methods of Treatment

This document provides methods and material for treating a mammal (e.g., a human) having a cancer (e.g., any appropriate cancer described herein) by administering therapeutically effective amounts of one or more (e.g., one, two, three, four, or more) EGFR inhibitors and one or more (e.g., one, two, three, four, or more) DDR1 inhibitors, to thereby treat the cancer in the mammal.

Examples of EGFR inhibitors that can be used as described herein include, without limitation, Lapatinib, Afatinib, Sapitinib, Erlotinib, and Gefitinib. Any appropriate EGFR polypeptide can be inhibited using an EGFR inhibitor described herein. For example, a human EGFR polypeptide having the amino acid sequence set forth in GenBank Accession No. NP_005219 (version NP_005219.2) can be the target of inhibition by an EGFR inhibitor described herein.

Examples of DDR1 inhibitors that can be used as described herein include, without limitation, small molecule DDR1 inhibitors, anti-DDRl antibodies, and nucleic acid-based inhibitors of DDR1 polypeptide expression (e.g., RNAi molecules). In some cases, a DDR1 inhibitor that can be used as described herein can be Bafetinib, Ponatinib, DDR1 7rh, VU6015929, Nilotinib, Sorafenib, PLX8394, Dactolisib, Imatinib, Dasatinib, DDR1-IN-1 (Kim et al., ACS Chemical Biology, 8(10):2145-2150 (2013)), Merestinib, or RAF709 (Nishiguchi et al., J. Med Chem., 60(12):4869-4881 (2017)). Any appropriate DDR1 polypeptide can be inhibited using a DDR1 inhibitor described herein. For example, a human DDR1 polypeptide having the amino acid sequence set forth in GenBank Accession No. NP 001284583 (version NP 001284583.1) can be the target of inhibition by a DDR1 inhibitor described herein.

An EGFR inhibitor can be an inhibitor of EGFR polypeptide activity or an inhibitor of EGFR polypeptide expression. Examples of compounds that can reduce or eliminate EGFR polypeptide activity include, without limitation, anti-EGFR antibodies and small molecules that target (e.g., target and bind) to an EGFR polypeptide. A DDR1 inhibitor can be an inhibitor of DDR1 polypeptide activity or an inhibitor of DDR1 polypeptide expression. Examples of compounds that can reduce or eliminate DDR1 polypeptide activity include, without limitation, anti-DDRl antibodies and small molecules that target (e.g., target and bind) to a DDR1 polypeptide.

Administration of one or more (e.g., one, two, three, four, or more) EGFR inhibitors and one or more (e.g., one, two, three, four, or more) DDR1 inhibitors can be effective to reduce or eliminate the level of EGFR polypeptides in a cancer cell (e.g., a cancer cell in a mammal such as a human). A reduced level of EGFR polypeptides refers to any level of EGFR polypeptides that is lower than the level of EGFR polypeptides typically observed in cancer cells prior to administration. An eliminated level of EGFR polypeptides refers to any non-detectable level of EGFR polypeptides. Any appropriate method can be used to determine whether or not a cell has a reduced or eliminated level EGFR polypeptides. For example, western blotting, reversetranscription polymerase chain reaction (RT-PCR), spectrometry methods (e.g., high- performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC/MS)), enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, immunofluorescence microscopy, CO-Detection by indexing (CODEX) imaging, and/or mass cytometry (CyTOF) can be used to determine whether or not a cell contains a reduced or eliminated levels of EGFR polypeptides. For example, administering one or more (e.g., one, two, three, four, or more) EGFR inhibitors in combination with one or more (e.g., one, two, three, four, or more) DDR1 inhibitors can be effective to reduce the expression level of EGFR polypeptides in a cancer cell (e.g., a cancer cell in a mammal such as a human) by, for example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more.

One or more (e.g., one, two, three, four, or more) EGFR inhibitors and one or more (e.g., one, two, three, four, or more) DDR1 inhibitors can be administered to a mammal having a cancer at the same time or independently. When one or more (e.g., one, two, three, four, or more) EGFR inhibitors and one or more (e.g., one, two, three, four, or more) DDR1 inhibitors are administered at the same time, one or more (e.g., one, two, three, four, or more) EGFR inhibitors and one or more (e.g., one, two, three, four, or more) DRR1 inhibitors can be administered as separate compositions administered at the same time or can be present in a single composition.

In some cases, the ratio of the one or more (e.g., one, two, three, four, or more) EGFR inhibitor(s) administered to a mammal (e.g., a human) to the one or more (e.g., one, two, three, four, or more) DDR1 inhibitor(s) administered to the mammal can be about 5: 1, about 4:1, about 3: 1, about 2: 1, about 1 : 1, about 1 :2, about 1 :3, about 1 :4, about 1 :5, or any sub-range therein. For example, in some cases, the EGFR inhibitor can be Lapatinib, the DDR1 inhibitor can be PLX8394, and the ratio of Lapatinib to PLX8394 administered to a mammal can be about 2: 1. In other cases, the EGFR inhibitor can be Lapatinib, the DDR1 inhibitor can be Bafetinib, and the ratio of Lapatinib to Bafetinib administered to a mammal can be about 1 : 1 or about 1 :2. In other cases, the EGFR inhibitor can be Lapatinib, the DDR1 inhibitor can be Ponatinib, and the ratio of Lapatinib to Ponatinib administered to a mammal can be about 2:1 or about 3 :1. In other cases, the EGFR inhibitor can be Lapatinib, the DDR1 inhibitor can be DDR1 7rh, and the ratio of Lapatinib to DDR1 7rh administered to a mammal can be about 2: 1. In still other cases, the EGFR inhibitor can be Lapatinib, the DDR1 inhibitor can be Nilotinib, and the ratio of Lapatinib to Nilotinib administered to a mammal can be about 1 : 1. In some cases, one or more EGFR (e.g., one, two, three, four, or more) inhibitors and one or more (e.g., one, two, three, four, or more) DDR1 inhibitors can be administered to a mammal having a cancer as the sole active ingredients used to treat a cancer.

In some cases, one or more (e.g., one, two, three, four, or more) EGFR inhibitors and one or more (e.g., one, two, three, four, or more) DDR1 inhibitors can be administered to a mammal having a cancer as a combination therapy with one or more additional cancer treatments used to treat a cancer. For example, a combination therapy used to treat a cancer can include administering to the mammal (e.g., a human) one or more (e.g., one, two, three, four, or more) EGFR inhibitors and one or more (e.g., one, two, three, four, or more) DDR1 inhibitors described herein and one or more cancer treatments such as surgery, chemotherapy, radiation, targeted therapy (e.g., a Src inhibitor, a BCR inhibitor, an ABL/ABL-1 inhibitor, a PI3K/mTOR inhibitor, a RAF inhibitor, an ERK1/2 inhibitor, a MEK inhibitor, a HDAC inhibitor, or a Notch inhibitor), and/or immunotherapy. In cases where one or more (e.g., one, two, three, four, or more) EGFR inhibitors and one or more (e.g., one, two, three, four, or more) DDR1 inhibitors described herein are used in combination with one or more additional cancer treatments, the one or more additional cancer treatments can be administered at the same time or independently. For example, one or more (e.g., one, two, three, four, or more) EGFR inhibitors and one or more (e.g., one, two, three, four, or more) DDR1 inhibitors described herein can be administered first, and the one or more additional cancer treatments can be administered second, or vice versa.

In some cases, an EGFR inhibitor and/or a DDR1 inhibitor administered to a mammal to treat cancer as described herein can target at least one of a Src kinase, BCR, and ABL/ABL-1. In some cases, an EGFR inhibitor and/or a DDR1 inhibitor administered to a mammal to treat cancer as described herein can inhibit MAPK signaling downstream of KRAS.

In some cases, a method provided herein can include administering one or more (e.g., one, two, three, four, or more) Src inhibitors (e.g., KB SRC-4). In some cases, a method provided herein can include administering one or more (e.g., one, two, three, four, or more) degraders of BCR-ABL (e.g., GMB475).

Cancer

The methods and materials described herein can be used to treat any appropriate cancer. Non-limiting examples of cancers that can be treated as described herein include: acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, brain tumor, bile duct cancer, bladder cancer, bone cancer, breast cancer, bronchial tumor, Burkitt lymphoma, cervical cancer, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloproliferative neoplasm, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T- cell lymphoma, ductal carcinoma, embryonal tumor, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, fibrous histiocytoma, Ewing sarcoma, eye cancer, germ cell tumor, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic disease, glioma, head and neck cancer, hairy cell leukemia, hepatocellular cancer, histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor, Kaposi sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cavity cancer, liver cancer, lobular carcinoma in situ, lung cancer, lymphoma, macroglobulinemia, malignant fibrous histiocytoma, melanoma, Merkel cell carcinoma, mesothelioma, mouth cancer, multiple myeloma, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer, penile cancer, pituitary tumor, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell cancer, renal pelvis and ureter cancer, retinoblastoma, rhabdoid tumor, salivary gland cancer, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cord tumor, stomach cancer, T-cell lymphoma, teratoid tumor, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, and Wilms’ tumor.

In some cases, the methods and materials described herein can be used to treat a cancer such as a colorectal cancer, brain cancer (including glioblastoma such as a glioblastoma multiforme), lung cancer, pancreatic cancer, or breast cancer.

In some cases, a cancer that can be treated as described herein can be a cancer having a KRAS mutation (e.g., a KRAS G13D mutation, a KRAS G12V mutation, a KRAS G12A mutation, a KRAS G12C mutation, a KRAS G12D mutation, a KRAS G12R mutation, a KRAS Q61L mutation, a KRAS Q61K mutation, a KRAS Q61R mutation, a KRAS Q61 V mutation, a KRAS Q61 A mutation, or a KRAS Q61C mutation). In some cases, a cancer that can be treated as described herein can be an EGFR inhibitor refractory cancer. In some cases, a cancer that can be treated as described herein can be an EGFR inhibitor resistant cancer (e.g., a Cetuximabresistant cancer). In some cases, a cancer that can be treated as described herein can be a chemo- radio-resistant cancer.

In some cases, a mammal having cancer that can be treated as described herein can be a mammal that was previously treated with a cancer treatment (e.g., at least one cancer treatment) for that cancer. Examples of previous cancer treatment that the mammal (e.g., human) could have received include, without limitation, one or more prior EGFR inhibitor treatments and/or one or more prior chemotherapies.

Any appropriate method can be used to identify a mammal (e.g., a human) having a cancer to be treated as described herein. For example, blood tests (e.g., complete blood count (CBC) tests, circulating tumor cell tests, tumor marker tests, and blood protein testing), imaging techniques (e.g., ultrasound, computerized tomography (CT) scanning, and magnetic resonance imaging (MRI)), and/or tissue biopsy techniques can be used to identify mammals (e.g., humans) having a cancer to be treated as described herein.

Once identified as having a cancer, a mammal (e.g., a human) can be administered, or instructed to self-administer, one or more (e.g., one, two, three, four, or more) EGFR inhibitors and one or more (e.g., one, two, three, four, or more) DDR1 inhibitors.

Any appropriate method can be used to identify a mammal as having, or as being at risk of developing, a cancer that exhibits little or no response to treatment with an EGFR inhibitor alone. In some cases, the over-expression or under-expression of one or more polypeptides in a sample can be used to identify the mammal as having, or as being at risk of developing, a cancer that exhibits little or no response to treatment with an EGFR inhibitor alone. Any appropriate sample can be used to detect the presence or absence of one or more polypeptides, or the presence or absence of a nucleic acid (e.g., a mutated genomic or a mRNA sequence). In some cases, a sample can be a biological sample. In some cases, a sample can contain one or more (e.g., one, two, three, four, or more) cells. In some cases, a sample can contain one or more (e.g., one, two, three, four, or more) biological molecules (e.g., nucleic acids such as DNA and RNA, polypeptides, carbohydrates, lipids, hormones, and/or metabolites).

Examples of samples that can be obtained from a mammal and used to detect the presence or absence of one or more polypeptides or the presence or absence of one or more nucleic acids include, without limitation, tissue samples (e.g., a cancerous tissue obtained from any of the cancers described herein), fluid samples (e.g., whole blood, serum, plasma, urine, and saliva), and cellular samples (e.g., nasopharyngeal samples, and buccal samples). A sample can be a fresh sample or a fixed sample (e.g., a formaldehyde-fixed sample or a formalin-fixed sample). In some cases, a sample can be a processed sample (e.g., an embedded sample such as a paraffin or OCT embedded sample). In some cases, one or more (e.g., one, two, three, four, or more) biological molecules can be isolated from a sample. For example, nucleic acids (e.g., DNA and RNA such as messenger RNA (mRNA)) can be isolated from a sample and can be used to detect the presence or absence of a nucleic acid. For example, one or more (e.g., one, two, three, four, or more) polypeptides can be isolated from a sample and can be used to detect the presence or absence of one or more polypeptides. Any appropriate method can be used to detect the presence or absence of a nucleic acid or one or more polypeptides. In some cases, polymerase chain reaction (PCR)-based techniques such as quantitative reverse transcription (RT)-PCR (qPCR) techniques, RNA in situ hybridization (ISH), and/or nucleic acid (e.g., DNA or RNA) sequencing can be used to detect the presence or absence of a nucleic acid. In some cases, immunoassays (e.g., immunohistochemistry (IHC) techniques, and western blotting techniques), mass spectrometry techniques (e.g., proteomics-based mass spectrometry assays or targeted quantification-based mass spectrometry assays), and/or enzyme-linked immunosorbent assays (ELISAs) can be used to detect the presence or absence of one or more polypeptides.

Pharmaceutical Compositions and Kits

Also provided herein are pharmaceutical compositions that include one or more (e.g., one, two, three, four, or more) EGFR inhibitors (e.g., one or more of any of the EGFR inhibitors described herein) and one or more (e.g., one, two, three, four, or more) DDR1 inhibitors (e.g., one or more of any of the DDR1 inhibitors described herein).

A pharmaceutical composition provided herein can be formulated to be compatible with their intended route of administration (e.g., an intravenous, subcutaneous, intraperitoneal, rectal, or oral route of administration). In some cases, a pharmaceutical composition provided herein can include a pharmaceutically acceptable carrier (e.g., phosphate buffered saline). Single or multiple administrations of formulations can be given depending on, for example, the dosage and frequency required and tolerated by the mammal (e.g., the human). The dosage, frequency, and timing required to effectively treat a mammal (e.g., a human) may be influenced by the age of the mammal, the general health of the mammal, the severity of the disease, previous treatments, and the presence of comorbidities (e.g., diabetes). The formulation can be designed to provide a sufficient quantity of active agents to effectively treat, prevent, or ameliorate conditions, diseases, or symptoms. Toxicity and therapeutic efficacy of compositions can be determined using procedures in cell cultures, pre-clinical models (e.g., mice, rats, or monkeys), and humans. Data obtained from in vitro assays and pre-clinical studies can be used to formulate the appropriate dosage of any composition described herein (e.g., any of the pharmaceutical compositions described herein).

In some cases, one or more (e.g., one, two, three, four, or more) EGFR inhibitors and/or one or more (e.g., one, two, three, four, or more) DDR1 inhibitors can be formulated into a composition (e.g., a pharmaceutically acceptable composition) for administration to a mammal having a cancer. For example, a therapeutically effective amount of one or more (e.g., one, two, three, four, or more) EGFR inhibitors and/or one or more (e.g., one, two, three, four, or more) DDR1 inhibitors can be formulated together with one or more pharmaceutically acceptable carriers (additives), excipients and/or diluents. A pharmaceutical composition can be formulated for administration in any appropriate dosage form. Examples of dosage forms include solid or liquid forms including, without limitation, gums, capsules, tablets (e.g., chewable tablets, and enteric coated tables), suppository, liquid, enemas, suspensions, solutions (e.g., sterile solutions), sustained-release formulations, delayed-release formulations, pills, powders, gels, creams, ointments, and granules.

Examples of pharmaceutically acceptable carriers, excipients, and diluents that can be used in a composition described herein include, without limitation, cyclodextrins (e.g., betacyclodextrins such as KLEPTOSE®), dimethylsulfoxide (DMSO), sucrose, lactose, starch (e.g., starch glycolate), cellulose, cellulose derivatives (e.g., modified celluloses such as microcrystalline cellulose, and cellulose ethers like hydroxypropyl cellulose (HPC) and cellulose ether hydroxypropyl methylcellulose (HPMC)), xylitol, sorbitol, mannitol, gelatin, polymers (e.g., polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), crosslinked polyvinylpyrrolidone (crospovidone), carboxymethyl cellulose, polyethylene-polyoxypropylene-block polymers, and crosslinked sodium carboxymethyl cellulose (croscarmellose sodium)), titanium oxide, azo dyes, silica gel, fumed silica, talc, magnesium carbonate, vegetable stearin, magnesium stearate, aluminum stearate, stearic acid, antioxidants (e.g., vitamin A, vitamin E, vitamin C, retinyl palmitate, and selenium), citric acid, sodium citrate, parabens (e.g., methyl paraben and propyl paraben), petrolatum, dimethyl sulfoxide, mineral oil, serum proteins (e.g., human serum albumin), glycine, sorbic acid, potassium sorbate, water, salts or electrolytes (e.g., saline such as phosphate buffered saline, protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, and zinc salts), colloidal silica, magnesium trisilicate, polyacrylates, waxes, wool fat, lecithin, and corn oil.

A composition (e.g., a pharmaceutical composition) containing one or more (e.g., one, two, three, four, or more) EGFR inhibitor and/or one or more (e.g., one, two, three, four, or more) DDR1 inhibitors can be designed for oral or parenteral (including, without limitation, subcutaneous, intratumoral, intramuscular, intravenous, topical, intradermal, intra-cerebral, intrathecal, or intraperitoneal injection) administration to the mammal. Compositions suitable for parenteral administration include, without limitation, aqueous and non-aqueous sterile injection solutions that can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient. The formulations can be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.

A composition (e.g., a pharmaceutical composition) containing one or more (e.g., one, two, three, four, or more) EGFR inhibitors and one or more (e.g., one, two, three, four, or more) DDR1 inhibitors can be administered locally or systemically. For example, a composition containing one or more (e.g., one, two, three, four, or more) EGFR inhibitors and/or one or more (e.g., one, two, three, four, or more) DDR1 inhibitors can be administered systemically by an oral administration or by injection to a mammal (e.g. a human).

Effective doses of one or more (e.g., one, two, three, four, or more) EGFR inhibitors and/or one or more (e.g., one, two, three, four, or more) DDR1 inhibitors can vary depending on the severity of the cancer, the route of administration, the age and general health condition of the subject, excipient usage, the possibility of co-usage with other therapeutic treatments such as use of other agents, and/or the judgment of the treating physician.

An effective amount of a composition containing one or more (e.g., one, two, three, four, or more) EGFR inhibitors and/or one or more (e.g., one, two, three, four, or more) DDR1 inhibitors can be any amount that can treat the cancer without producing significant toxicity to the mammal. An effective amount of an EGFR inhibitor can be any appropriate amount. In some cases, an effective amount of an EGFR inhibitor can be from about 0.1 mg/kg body weight of a mammal to about 30 mg/kg body weight of a mammal (e.g., from about 0.5 mg/kg to about 30 mg/kg, from about 0.75 mg/kg to about 30 mg/kg, from about 1 mg/kg to about 30 mg/kg, from about 1.5 mg/kg to about 30 mg/kg, from about 2.5 mg/kg to about 30 mg/kg, from about 5 mg/kg to about 30 mg/kg, from about 10 mg/kg to about 30 mg/kg, from about 20 mg/kg to about 30 mg/kg, from about 0.1 mg/kg to about 25 mg/kg, from about 0.1 mg/kg to about 20 mg/kg, from about 0.1 mg/kg to about 15 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 5 mg/kg, from about 0.1 mg/kg to about 1 mg/kg, from about 1 mg/kg to about 20 mg/kg, from about 5 mg/kg to about 15 mg/kg, or from about 7.5 mg/kg to about 12.5 mg/kg). An effective amount of a DDR1 inhibitor can be any appropriate amount. In some cases, an effective amount of a DDR1 inhibitor can be from about 1 mg/kg body weight of a mammal to about 30 mg/kg body weight of a mammal (e.g., from about 2 mg/kg to about 30 mg/kg, from about 3 mg/kg to about 30 mg/kg, from about 4 mg/kg to about 30 mg/kg, from about 5 mg/kg to about 30 mg/kg, from about 6 mg/kg to about 30 mg/kg, from about 7 mg/kg to about 30 mg/kg, from about 10 mg/kg to about 30 mg/kg, from about 20 mg/kg to about 30 mg/kg, from about 1 mg/kg to about 25 mg/kg, from about 1 mg/kg to about 20 mg/kg, from about 1 mg/kg to about 15 mg/kg, from about 1 mg/kg to about 10 mg/kg, from about 1 mg/kg to about 5 mg/kg, from about 1 mg/kg to about 1.5 mg/kg, from about 1 mg/kg to about 15 mg/kg, from about 5 mg/kg to about 15 mg/kg, or from about 7.5 mg/kg to about 12.5 mg/kg).

The effective amount can remain constant or can be adjusted as a sliding scale or variable dose depending on the mammal’s response to treatment. Various factors can influence the actual effective amount used for a particular application. For example, the frequency of administration, duration of treatment, use of multiple treatment agents, route of administration, and severity of the condition (e.g., a cancer) may require an increase or decrease in the actual effective amount administered.

The frequency of administration of a composition containing one or more (e.g., one, two, three, four, or more) EGFR inhibitors and/or one or more (e.g., one, two, three, four, or more) DDR1 inhibitors can be any frequency that can treat the cancer without producing significant toxicity to the mammal. For example, the frequency of administration can be from about three times a day to about once a week, from about twice a day to about twice a week, or from about one a day to about twice a week. The frequency of administration can remain constant or can be variable during the duration of treatment. A course of treatment with a composition containing one or more (e.g., one, two, three, four, or more) EGFR inhibitor and/or one more (e.g., one, two, three, four, or more) DDR1 inhibitors can include rest periods. For example, a composition containing one or more (e.g., one, two, three, four, or more) EGFR inhibitors and/or one or more (e.g., one, two, three, four, or more) DDR1 inhibitors can be administered daily over a two-week period followed by a two-week rest period, and such a regimen can be repeated multiple times. As with the effective amount, various factors can influence the actual frequency of administration used for a particular application. For example, the effective amount, duration of treatment, use of multiple treatment agents, route of administration, and severity of the condition (e.g., a cancer) may require an increase or decrease in administration frequency.

An effective duration for administering a composition containing one or more (e.g., one, two, three, four, or more) EGFR inhibitors and/or one or more (e.g., one, two, three, four, or more) DDR1 inhibitors can be any duration that treat the cancer without producing significant toxicity to the mammal. For example, the effective duration can vary from several days to several weeks, months, or years. In some cases, the effective duration for the treatment of a cancer can range in duration from about one month to about 10 years. Multiple factors can include the actual effective duration used for a particular treatment. For example, an effective duration can vary with the frequency of administration, effective amount, use of multiple treatment agents, route of administration, and severity of the condition being treated.

In some cases, the number of cancer cells present within a mammal and/or the severity of one or more symptoms of the cancer being treated can be monitored. Any appropriate method can be used to determine whether or not the number of cancer cells present within a mammal is reduced. For example, imaging techniques can be used to assess the number of cancer cells present within a mammal before, after, and/or during treatment as described herein.

Efficacy of any of the compositions described herein can be determined, for example, by the observation of the clinical signs of a cancer (e.g., tumor size, presence of metastasis).

Also provided herein are kits that include one or more (e.g., one, two, three, four, or more) EGFR inhibitors (e.g., one or more of any of the EGFR inhibitors described herein) and one or more (e.g., one, two, three, four, or more) DDR1 inhibitors (e.g., one or more of any of the DDR1 inhibitors described herein). In some cases, the kits can include at least one dose of any of the pharmaceutical compositions described herein.

The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLES

Example 1: Targeting DDR1 /BCR-ABL with EGFR-ERBB2/3 Against Chemo-Radio-Resistance

Methods

Selection of small molecule Inhibitor panel

A panel of 38 inhibitors were selected against various signaling pathway intermediates (Tables 1 and 2). 33 inhibitors were used in the initial screening, of which 10 inhibitors were registered FDA approved drugs, 9 inhibitors were investigational drugs in clinical trials, and the remaining 14 inhibitors were drugs under preclinical testing, or tool compounds. Additionally, inhibitors that were incorporated in the study included, nilotinib (FDA approved), dasatinib (FDA approved), imatinib (FDA approved), KB-SRC-4 (preclinical Src kinase inhibitor), and GMB475 (BCR-ABL, a proteolysis-targeting chimera (PROTAC)). All inhibitors were dissolved in sterile DMSO (Sigma- Aldrich) and stored frozen at -20 degrees Celsius in aliquots to avoid repeated freeze-thaw. A maximal concentration of 0.1% DMSO was used as control.

Table 1. List of Inhibitors

Table 2. Drug Screen using HCT116 cells.

Cell culture

Human cell lines for colorectal adenocarcinoma (HCT116, DLD1, SW480, and CaCO2) and a glioblastoma cell line (U251) were purchased from the American Type Culture Collection (ATCC) or from Sigma Aldrich, expanded, and maintained in ATCC-recommended media as per standard cell culture practices. Patient derived glioblastoma cells (PD-GBM) GBM965 and QNS108 were isolated as described elsewhere (Garcia et al., Mol. Cancer Ther., 20(12):2585- 2597 (2021) and Wong et al., Nat. Biomed. Eng., 5(l):26-40 (2021)).

3D multi-spheroid cultures

Multi-spheroid 3D cultures were established by seeding cells at optimized densities between 4000-7000 cells/well in standard 96-well flat-bottom plates having Nunclon delta surface (167008, Thermofisher Scientific) in their respective culture media (Figure 9 and Table 3).

Table 3. Culture conditions used for multi-spheroid growth

The spheroids were cultivated in media having 1% penicillin/ streptomycin (15140122, Gibco), without 10% fetal bovine serum (FBS), and having additional supplements as indicated: N2 supplement (N-2 Max (100X), AR009, R&D systems), N21 (N21 Max (50X), AR008, R&D systems), recombinant human epidermal growth factor (EGF) (AF-100-15, PeproTech), recombinant human fibroblast growth factor 2 (FGF2) (AF-100-18B, PeproTech), insulin- transferrin-selenium-ethanolamine (ITSx (100X), 51500056, Gibco). Spheroid culture media having N2 supplement with EGF and FGF2 was defined as N2EF media; media with N2 supplement and N21max was defined as N2N21max media, and media having N2 supplement having EGF, FGF2 and ITSx was defined as N2EF+ITSx. Spheroid growth was measured using the following parameters: (i) increase in spheroid size over time and (ii) metabolic activity as an indicator of spheroid cell viability.

Spheroid growth was monitored utilizing the Incucyte® 3D Multi-Tumor Spheroid module installed in Incucyte® Live-Cell Imaging System (Incucyte SX3, Sartorious) that can evaluate label-free development of 3D spheroids in real time. The images obtained were masked or pseudo-colored by built-in Incucyte® image processing component. Change in spheroid total area, spheroid average area, and spheroid eccentricity were plotted over time as measures of spheroid development, growth, and circularity, to obtain breadth of information on spheroid formation and health rates pre- or post-drug treatments. Additionally, averaged spheroid size was estimated in bright field using Evos® FL microscope at 40x magnification (15 spheroids imaged per culture condition, and average diameter recorded). Metabolic activity was measured as a measure of increased spheroid size or spheroid cell viability over time using the luminescent-based cell adenosine nucleotide triphosphate (ATP) release assay CellTiter- Gio® (G7572, Promega). Viable cells were counted at the beginning of every experiment using a TC20 automated cell counter (BioRad).

Irradiation

Cells were cultured in spheroid growth media in standard 96-well plate for 24 hours. X- ray photon irradiation was administered by X-RAD® 160 X-Ray Biological Irradiator (Precision X-Ray Inc., North Branford, CT, USA) at max mA=18.7, max kV=160, and dose rate 403.8 cGy/min. After irradiation (IR) treatment, the spheroids were continued to be cultured until two time points: 24 hours post-IR and day 5 post-IR treatment. Spheroid growth and health was evaluated based on increase in reactive oxygen species (ROS) levels (ROS-Glo™ H2O2 Assay, G8820, Promega)), change in spheroid cell viability (CellTiter-Glo®, G7572, Promega), and induction of caspase 3/7 activity (Caspase-Gio® 3/7 Assay System, G8090, Promega). All multispheroid assays were performed as per manufacturer’s protocol (Figure 12C). To evaluate the effect of drugs on overcoming intrinsic radio-resistance and spheroid viability post-IR treatment, HCT1 16 spheroids were cultured for 24 hours, IR treated at doses 4 Gy, 8 Gy, 12 Gy, 16 Gy and 20 Gy, and the respective drugs were administered 24 hours post-IR with having DMSO as control. The spheroids were continued to be cultured until day 5-postIR, and spheroid health was estimated by change in spheroid cell viability, and induction of caspase 3/7 activity.

Multi-Spheroid drug screens Single drug testing

Exponentially growing HCT116 multi-spheroids cultured in N2EF media were administered with inhibitors serially diluted in DMEM (no FBS) at doses up to 25 pM in triplicates. After 2 days post-drug administration, the spheroid viability was measured (CellTiter-Glo, G7572, Promega). Single drug response curves were obtained similarly for the other colorectal adenocarcinoma (COAD) lines investigated. For glioblastoma multiforme (GBM) line U251, spheroids were obtained by day 6, drug treatments were performed and, measurements done by day 12. Glioblastoma multiforme-patient derived xenograft (GBM-PDX) lines (GBM965 and QI 08) were evaluated for intrinsic radio-sensitivity (Figure 8), and inhibitors were administered as single agents or in combination, to monitor their efficacy on spheroid development and growth. Data obtained for each treatment was normalized to DMSO control. Dose-response curves were generated and inhibitory dose 50 (IC50) values were determined using graph pad prism software (GraphPad, Inc.).

Combinatorial drug testing at single dose

To evaluate the drug interactions in single-dose assay, the drugs identified to have IC50 < 25 pM from single drug testing were combined with small molecule inhibitors for wild-type epidermal growth factor receptor (EGFR) (Lapatinib, Afatinib, and Sapitinib), at combination dose < their estimated IC50 values, using the experimental scheme, unless specified (Figure 9C). The treatments were done for two days for spheroids obtained from COAD lines, for five days in GBM (U251) spheroids and six days on GBM-PDX lines, and the combinatorial drug response was measured based on spheroid viability assay (CellTiter-Glo, G7572, Promega). All treatments were done in triplicates, and data was normalized to that of the DMSO control. Doses used for combination testing are in Tables 2, 4A, 4B, 4C, 5 A, 5B, 6, and 7.

Table 4 A. Doses of drugs used in combination treatments for COAD and GBM lines.

Key: NA: Not Applicable (when treatment not tested); NR: Not reachable (desired result not reached).

Table 4B. Spheroid viability assay performed for DDR1/BCR-ABL1 inhibitors in COAD and

GBM lines.

Table 4C. Spheroid viability assay performed at late time-points (i.e., treatments performed at late exponential phase of growth / oversized spheroids) in COAD and GBM lines.

Table 5A. Efficacy of drugs alone or in combination with EGFR small molecule inhibitors,

Lapatinib, Afatinib and Sapitinib, in HCT116 spheroids (cultured in N2EF media).

Table 5B. Average of percent (%) inhibition estimated for Drugs ± EGFRi in HCT116 and DLD1 spheroids (administered with Drugs + EGFRi, Lapatinib, Afatinib), and SW480 spheroids (administered with Drugs + EGFRi Lapatinib) during multispheroid assays.

Table 6. Inhibitor Combinations used in Synergy experiments for COAD and GBM.

Table 7. Inhibitor Combinations used in Synergy experiments for COAD and GBM (Continued).

Drug Synergy testing

To identify the interactions between two drugs and to estimate whether the effect was additive, synergistic, or antagonistic, the drugs were combined at five individual doses. These five doses for each drug were selected such that they were equi-proportionally spread across its estimated IC50 and follow their IC50 potency ratio. In a matrix of five doses of Drug- 1 and five doses of Drug-2, 5 (Drug-1) x 5 (Drug-2) = 25 different combinations were obtained and administered in quintuplicates on exponentially growing multi-spheroid cultures at conditions optimized for single-drug testing. For each drug, all five doses were also administered as single agent, along with DMSO as untreated control. The spheroid viability was measured, and data was computed into the MacSynergy™-!! software (Prichard el al. , Antiviral Res., 14(4-5): 181- 205 (1990)). Data obtained was represented in form of (i) 3D surface plots, where contours above the plane were indicative of synergy and depressions below the plane indicated antagonism, (ii) Dot plots, where spheroid viability for all doses of Drug- 1, and Drug-2 were presented against the 25 different combinations administered. Each dot represented an individual treatment condition, (iii) Bar graphs, for spheroid viability measurements done at the treatment dosage where synergy score was maximum.

Clonal cell proliferation assay

HCT116 cells were plated at optimized seeding density (600 cells/well) in a standard 96- well plate (167008, Thermofisher Scientific), in growth media McCoy’s 5A medium (16600082, Gibco), with 10% FBS and 1% penicillin/ streptomycin. Drugs were administered after 24 hours, and colony growth was monitored over time utilizing the Incucyte®’s built in Clonal dilution module. Percent cell confluence was estimated as measure of cell proliferation, and automated pseudocolored images of clonal growth were acquired at Day 0, Day 3 and Day 5 for all treatment conditions. CaCO2 cultures were evaluated similarly for clonal cell proliferation (Figure 18B).

To assess clonal cell growth and proliferation over time in PD-GBM, GBM965 and QNS108, the cells were seeded at a density of 1000 cells/well in a standard 96 well plate in stemcell media, (same as utilized for spheroid cultures, having DMEM-F12, supplemented with N2- Max plus, N21max, EGF, FGF2 and ITSx), with or without the presence of drug combinations being tested. The cells were allowed to grow for a week and then assessed for viability (CellTiter-Glo®, G7572, Promega).

Cancer Genomics and Bioinformatics

(i) Evaluation of percent genetic variations: COAD and GBM datasets from The Cancer Genome Atlas (TCGA) combined with all COAD or GBM studies deposited at cBioPortal database, and PanCancer Atlas were utilized to obtain percent genetic alterations for the selected gene list (source: cBioPortal).

(ii) Relative gene expression: The mRNA expression of the selected genes was evaluated and compared in two biological states: Normal tissue and tumor tissue, for both colorectal adenocarcinoma and glioblastoma, utilizing Gene expression profiling interactive analysis, gene expression profiling interactive analysis (GEPIA) that links the datasets from TCGA (for tumor tissue) and GTEx (for normal tissue)

(iii) Profiling of genes co-expressed with Discoidin domain receptor 1 (DDR1) and Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS): GEPIA was utilized to perform a pair- wise gene expression correlation analysis of genes DDR1 and KRAS with selective genes through TCGA and GTEx expression datasets using Pearson method. Additionally, Pan Cancer Atlas dataset (c-Bioportal) was utilized to perform a pair-wise gene expression correlation analysis using the Pearson and Spearman method.

(iv) Protein-interactions were evaluated utilizing the BioGRID and STRING databases.

(v) IC50 correlations for EGFR inhibitors and BCR-ABL1 inhibitors were obtained for colorectal adenocarcinoma (COREAD), and PanCancer cell line datasets using Genomics of Drug Sensitivity in Cancer (GDSC) database.

Statistical analysis

Data analysis was performed using Graphpad Prism and student’s t-test was utilized to obtain as statistical significance as indicated *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. All data is presented as the mean of minimally two independent experiments with error bars as standard error of mean (SEM).

Radio-sensitivity curves for QNS965 and QNS108

Radio- sensitivity curves for radio-resistant GBM lines were build as described in Lara et al., Cancer Letters, 365:5-16 (2015). Briefly, GBM cells seeded on sextuplicates on ultra-low attachment 96 well plates received a single dose of 0 Gy, 2 Gy, 4 Gy, or 6 Gy (275.1 cGy/min) with an X-rad® 1600 (Precision X-Ray). Plating efficiency was determined after 3 weeks considering the number of spheres with more than 100 pm of diameter. Surviving factors at each dose were then fitted to the linear quadractic model with R studio (R-Studio Team. RStudio: Integrated development environment for r. (RStudio, PBC, 2020)) using the CF Assay R package (Braselmann, H., CF Assay: Statistically analysis for the colony formation assay (2020)). Surviving fractions at 2 Gy (SF2) were used to determine sensitivity to radiation, GBM cell lines with SF2>40% were considered radioresistant (Figure 8). Results

Growth and expansion of multi-spheroids in defined media

HCT1 16 and DLD1 cell lines harboring KRASG13D mutation and, classified as C4 (Marisa class) and stem-like (Sadanandam class) were optimized for 3D multi-spheroid cultures using two different spheroid growth media, N2EF, and N2N21max. Both media conditions were found to be comparable for formation of proliferative HCT116 multi- spheroids (Figure 1 A, Figure 10). Thus, all experiments were carried in N2EF media for HCT116, unless indicated for N2N21max. DLD1 formed proliferative spheroids only in N2N21max supplemented media (Figure 3A, Figure 10). Evaluating the spheroid growth parameters measure using incucyte, exponential growth of spheroids was observed between day 1.5 and day 4.5 for both the lines (Figure 10D). Therefore, all drug screens were performed within this period (Scheme, Figure 9C). Spheroid culture parameters evaluated for SW480 (KRASG12V) showed similar trend with culture expansion best observed between dayl.4 to 4.5 (Figure 3C, Figure 10E). GBM lines were slower in growth relative to COAD lines investigated. U251 (GBM line) cells were optimized for multi- spheroid assays similarly, and spheroid formation was observed by day 6; therefore, drug treatments were performed between day 6 and day 12.

Treatment resistance and WNT effectors in multi-spheroids of COAD

HCT116 multi-spheroids exhibit intrinsic resistance to cetuximab and irradiation treatment Cetuximab administered up to a final concentration of 25 pg/well on HCT116 multispheroids cultured in N2EF and N2N21max, showed no cytotoxic effect indicating anti-EGFR resistance in HCT116 (Figure 1 A). To evaluate the effect of IR on spheroid survival and growth, HCT1 16 multi-spheroids were administered with single-dose radiations: 0 Gy (untreated control), 4 Gy, 8 Gy, 12 Gy, 16 Gy, and 20 Gy, respectively (Figure 9C). Minimal increase in caspase3/7 activity with concomitant decline in spheroid viability was observed, despite significant rise in ROS levels from 0 Gy to 20 Gy at 24 hours post-IR. However, irradiated spheroids that were continued to be cultured until day 5, showed significant decline in ROS levels, and increase in spheroid size at all irradiation treatments despite induction of intrinsic caspase3/7 activity by day5 post-IR. Increased spheroid viability at day 5 post-IR as compared to 24 hour post-IR is indicated (p-value < 0.0001) (Figure IB). To rule-out the effect of media composition, similar evaluation was performed on HCT116 multi-spheroids grown in N2N21max. The data-trend observed was same irrespective of culture conditions (Figure 1A-1B, Figure 12B), indicating resistance to cetuximab and irradiation as an intrinsic property of HCT116 cells.

Targeting WNT-effector f>-calenin reduced COAD multi-spheroid viability

Sternness in multi-spheroids of HCT116 (cultured in N2EF) was evaluated using small molecule inhibitors that could target WNT pathway at either the level of cell surface receptor (FZ 7-21, Salinomycin) or receptor folding (XAV939) or by inhibiting downstream WNT effectors (BC2059, PRI724). No effect was observed by receptor inhibition, but significant cytotoxicity in response to targeting P-catenin or P-catenin/P300 complex indicated constitutive activation of down-stream effectors in HCT116 (Figure ID). DLD1 multi-spheroids showed reduced viability upon targeting P-catenin or P-catenin/P300 as observed for HCT116 (Figure 3A).

Small molecule Inhibitor -based drug screening in HCT116 multi-spheroids

A drug screen performed utilizing a panel of 33 small molecule inhibitors (Table 1), on multi-spheroids from HCT116 cells revealed 21 effective inhibitors, in single agent doseresponse curves. These included small molecule inhibitors of EGFR, and selective compounds targeting WNT/p-catenin, PIK3/mT0R dual kinase, epigenetic regulators, RAF/BRAF, multityrosine kinases, and NTRK (neurotrophic receptor tyrosine kinases). Challenges to obtain KRAS mutation-specific drugs led to a poor response from direct KRAS targeting (Figures 1C and 9A). Inhibitory dose 50 (IC50) values estimated are included in Table 2. Dose response curves obtained for WNT inhibitors and EGFR small molecule inhibitors are included in Figure ID, and dose response curves for all other inhibitors tested are included in Figure 11.

Combinatorial drug testing at single dose

Investigating the efficacy of 19 inhibitors identified in combination with small molecule inhibitors for wild-type EGFR (EGFRi: Lapatinib, Afatinib and Sapitinib) revealed that 9 inhibitors exhibited enhanced cytotoxicity in combination as compared to as single-agents (Table 2). These included Paxalisib, Dactolisib, PLX8394, Bafetinib, Ponatinib, TPX0005, DDR17rh, VU6015929, and Vorinostat (Figure 12A) Sorafenib showed promising response with Lapatinib, and the remaining 9 inhibitors showed moderate to low effect in combination with EGFRi (Figure 12A). Viable drug combinations were identified based on two criteria: (a) if the percentage of inhibition obtained in combination with at least two out of three EGFRi was greater than or equal to 50%, and (The drugs that met the first criteria were Dactolisib, PLX8394, Bafetinib, Ponatinib, TPX0005, VU6015929, DDR1 7rh, and Vorinostat; and the 5 drugs that satisfied both the criteria, thus being our Top-5 candidates identified were Bafetinib, Ponatinib, VU6015929, DDR1 7rh and PLX8394 (Figure IE, and Tables 5A and 5B). Overall combinatorial response for all 3 EGFRi were in the order Lapatinib > Afatinib > Sapitinib.

These results were validated in HCT116 and DLD1 multi-spheroids (N2N21max cultured (Figure IB)). In HCT116, all 5 leads were more potent when coadministered with EGFRi (Lapatinib or Afatinib), and most potent when combined with Lapatinib (Figure 2). DLD1 is a colorectal adenocarcinoma cell line that, like HCT116, harbors heterozygous KRASG13D mutation and has intrinsically high WNT activity as confirmed by its response to inhibition of WNT effector, P-catenin (Figure 3 A). DLD1 multi-spheroids were affected by combination treatment with EGFRi similarly to HCT116 multi- spheroids. Percent inhibition obtained in combinations with lapatinib was greater than Afatinib (Figure 3B), and the leads showed potent combinatorial efficacy (Figure 1 IE). To test if these observations were KRAS mutation-type independent, combinatorial efficacy of drugs ± Lapatinib was validated in spheroid cultures of SW480, a COAD cell line homozygous for KRASG12V mutation (Figure 3C). The top-5 leads when administered in combination with EGFRi (lapatinib and afatinib) showed > 95% averaged percent inhibition for all three COAD lines tested, and, had an overall fold-change > 2 (Figure 3D). Bafetinib and ponatinib showed the best combinatorial response in all 3 lines, with averaged percent inhibition of > 95% and fold-change > 4 (Figure 3D). Drug doses used are included in Table 4.

Drug Synergy testing

Synergy was identified for five drugs (PLX8394, Bafetinib, Ponatinib, VU6015929, and DDR1 7rh) in combination with both EGFRi (Lapatinib and Afatinib) (Figure 4). When administered in combination with Lapatinib, drugs that showed peak synergy score > 100 (indicating strong synergy) were Bafetinib and PLX8394, and within 50-100 (indicating moderate but significant synergy) were Ponatinib = DDR1 7RH > VU6015929. When administered in combination with Afatinib, none of the drugs showed a peak synergy score > 100 and only drugs that showed peak synergy between 50-100 were Ponatinib and PLX8394. A widespread rise in 3D surface-plot observed for Lapatinib plus Bafetinib indicated it to have highest synergy volume (1123.4). Comparing the cumulative synergy scores (volume of synergy) obtained for all 5 drugs, revealed combinations with lapatinib to have an overall higher synergy, and in order of Bafetinib > PLX8394 = Ponatinib > DDR1 7RH > VU6015929. Thus, EGFR/ERBB2 inhibitor Lapatinib was identified to be a more promising combinatorial drug in COAD. The matrix showing synergy scores and percent inhibitions obtained at each of the individual 25 combinations administered per synergy evaluation for all 5 drugs (in combination with lapatinib and afatinib) are included in Figure 13 and Figure 14 (dot plots and bar-graphs for spheroid viability are in Figure 15). Table 8 provides (a) the cumulative synergy- and antagonism- scores obtained for all combinations tested (drugs with Lapatinib or Afatinib) and (b) the doses of Drug- 1 and Drug-2 at which synergy was maximum and maximum synergy score value for each of the combinations tested.

Table 8. Synergy scores obtained in HCT116 multi-spheroid viability assay DDR1, a common target among synergistic leads identified

To address why these 5 inhibitors selectively emerged as potential synergistic leads, their common targets were examined. These were found to be: 1) DDR1 (Discoidin domain receptor 1), 2) BCR-ABL kinases 3) Src kinases (Src/Lyn) (Figure 4B). Bafetinib and Ponatinib belong to BCR-ABL family of multi-tyrosine kinase inhibitors which also target DDR1 (Holland et al., Nat. Genet., 25(1): 55-7 (2000)), and DDR1 was a common target among 4 out of top-5 combinatorial leads.

Cancer Genomics

To further evaluate these five inhibitors and their role in providing a synergistic response, their common targets were studied. These common targets were found to be: (1) Discoidin domain receptor 1 (DDR1), (2) BCR/ABL kinases, and (3) V-Src Avian Sarcoma (Schmidy- Ruppin A-2) Viral Oncogene Homolog (Src) kinases (Src/ Lck/Yes Related Novel Protein Tyrosine Kinase) (Src/Lyn) (Figure 4L). To investigate how their signaling cross-talks may contribute to disease progression, a bioinformatics approach was taken. A panel of 35 genes was selected incorporating direct or indirect targets of the identified combinatorial drugs (PLX8394, Bafetinib, Ponatinib, DDR1 7RH, VU6015929, Lapatinib and Afatinib), related biological processes (cell adhesion, proliferation, migration, Sternness) and genes known to be commonly mutated in colorectal cancer. DDR1 was a target for at least 4 out of 5 of the synergistic drugs identified. Thus, its relevance in COAD was investigated. DDR1 also was reported to be overexpressed in several tumors with maximal expression in brain (normal cortex, tumors). A parallel comparison of the selected gene panel was performed for the percentage of genomic alterations, relative mRNA expression, and transcriptional correlations, in datasets of COAD, GBM and PanCancer Atlas (Genomics portal: cBioPortal, and GEPIA). KRAS was included in all transcriptional correlations to identify common associations between DDR1 and KRAS signaling.

KRAS, a tumor driver in KRASmut COAD and GBM

Percent genetic alterations identified were adenomatous polyposis coli (APC) (63%), Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) (38%), Phosphatidylinositol-4,5- bisphosphate 3-kinase catalytic subunit-a (PIK3CA) (18%), V-Raf Murine Sarcoma Viral Oncogene Homolog Bl (BRAF) (11%), MTOR (6%), V-Src Avian Sarcoma (Schmidy-Ruppin A-2) Viral Oncogene Homolog (SRC) (5%), Catenin-Betal gene (CTNNB1) (6%), V-Erb-B2 Erythoblastic leukemia viral oncogene homolog 2/4 (ERBB2/4) (6%), V-Erb-B2 Erythoblastic leukemia viral oncogene homolog 3/ Epidermal growth factor receptor (ERBB3/EGFR) (4%), and, to lower extent in genes related to SRC/BCR pathway (ABL1/2, BCR, LYN, DDR1) and cell adhesion (DDR1, Cadherin-1 (CDH1), CTNNA1). Sternness related genes found to be altered were SRY-Box transcription Factor 9 (SOX9) > CTNNB1 » Van Gogh-Like Planar cell polarity protein 2 (VANGL2), SOX2, and SOX8. COAD was clearly a APC ^mut and KRAS ^mut driven tumor, as opposed to GBM having maximal percent alterations in EGFR (Source: cBioPortal). See, e.g., Figure 20, Table A, and Table B.

Table A.

Genomic associations of DDR1 with BCR-ABL, EGFR/ERBB, KRAS and sternness mRNA expression: At the transcriptional level, genes upregulated in COAD as compared to colon (normal tissue) were as follows: ERBB2, DDR1, BCR, SRC, LYN, Leucin Rich Repeat containing G-protein coupled receptor - 5 (LGR5) (WNT regulator), and CTNNB1 (P catenin). Expression of DDR1, EGFR, ABL1, and LYN was higher in GBM, with DDR1 expression being the highest (Genomics interface: GEPIA (Gene Expression Profiling Interactive Analysis), Datasets: Cancer Genome Atlas, TCGA (tumor), and Genotype-Tissue Expression, GTEx (Normal) (Figure 17A).

Correlation analysis: When evaluating for correlations between the mRNA expression of a panel of 33 genes, with mRNA expression of DDR1 and KRAS in datasets of COAD, GBM and PanCancer Atlas, EGFR, IGF1R, ERBB3, PIK3CA, SRC, BCR, ABL1, APC, and CDH1 were shown to correlate with DDR1 in COAD. ERBB3, SRC and CDH1 expression associated with DDR1 gene expression in both colon (normal tissue) and in COAD, and, EGFR, IGF1R, BCR, ABL1 and APC correlated with DDR1 expression exclusively in COAD. Expression of genes EGFR, ERBB4, BCR, SOX2, SOX9, and VANGL2 positively correlated with expression of DDR1 and KRAS in PanCancer dataset; and, EGFR, IGF1R, BRAF, PIK3CA, MTOR, BCR, ABL1, SOX8, APC and VANGL2 correlated with DDR1 in COAD (not, colon-normal). Expression of genes ERBB4, VEGFR1, KRAS, BRAF, PIK3CA, MTOR, SRC, BCR, APC, CTNNB1 and, CDH2 to correlate with DDR1 in GBM-dataset (not brain cortex (normal)). Thus, DDR1 positively correlated with BCR and, downstream mediators of KRAS signaling (BRAF, PIK3CA, MTOR) in both tumors, COAD and GBM. DDR1 correlates with genes EGFR, ERBB2, ERBB4, BRAF, BCR, and genes of sternness, polarity and adhesion (SOX9, VANGL2, CDH1) in PanCancer, with an overall positive association with expression of genes BRAF, BCR, and APC in tumors (Figure 5A). Evaluating the correlations between mRNA expression of the selected gene panel with the expression of KRAS, revealed positive correlation between KRAS and the genes BRAF, PIK3CA, APC and CTNNB1 in PanCancer (Figure 17B). BRAF, PIK3CA, MTOR, ABL1, and VANGL2 correlated with both DDR1 and KRAS in COAD, further indicating the implications of DDR1 -BCR-ABL 1 axis in activation of KRAS and PI3K/mT0R pathway. All correlation coefficients (Pearson, Rp or Spearman, Rs) > 0.2 were significant positive associations, (iv) To further investigate association of DDR1 with SRC/BCR- ABL signaling in COAD vs. colon (normal), a correlation matrix with selective genes was generated: EGFR, DDR1, SRC, LYN, BCR, LGR5, and CDH1. Significant correlations were observed between the expression of DDR1 and genes EGFR, and intracellular SRC/BCR axis indicating its active involvement in COAD (Source: GEPIA; datasets, TCGA (tumor), GTEx (Normal)) (Figure 17C and 17D).

Targeting DDR1/BCR-ABL signaling

To test whether the expectancy of an enhanced cytotoxic response like observed with DDR1/BCR-ABL multi-tyrosine kinase inhibitors (BCR- ABL MTK), Bafetinib and ponatinib, when administered in combination with EGFR inhibitor lapatinib could be extrapolated to also other members of DDR1/BCR-ABL inhibitor family, the drugs, Dasatinib, Imatinib and Nilotinib were incorporated into the evaluation. Nilotinib was observed to be more potent than Dasatinib and Imatinib as a single agent, and significant reduction in HCT116 spheroid viability was obtained with all three inhibitors Dasatinib, Imatinib and Nilotinib when administered in combination with Lapatinib (L) or Afatinib (A) (Figure 6 A). To identify whether combinatorial targeting of EGFR-ERBB2/4 with Src kinase or BCR- ABL 1 kinase alone could have similar outcome as observed with multi-tyrosine kinase inhibitors targeting DDR1/BCR-ABL1 signaling axis, two compounds were tested, KB SRC-4 (Src inhibitor), and GMB475 (PROTAC degrader of BCR-ABL). Both compounds showed enhanced response in combination with Lapatinib and Afatinib, with BCR-ABL specific targeting having a better efficacy than Src targeting (Figure 6A). Comparing the intrinsic caspase3/7 activity elicited by drug administration revealed much higher apoptosis induced in combinations with Lapatinib than Afatinib, indicating EGFR/ERBB2 targeting to be a more potent combination with DDR1 /BCR-ABL 1, as compared to targeting EGFR/ERBB4 (Figure 18 A).

Signaling interactome of DDR1 /BCR-ABL 1 :

To investigate the interconnections of DDR1, BCR, and ABL1 with KRAS signaling, SRC kinases (SRC/LYN), WNT effectors (CTNNB1, SOX factors), and EGFR signaling, protein-interaction analyses using BioGRID and STRING databases were performed. ERBB2 and CDH1 were found as close associates of DDR1 in BioGRID. STRING protein interactome for physical interactions between 32 proteins (32 nodes and 112 edges) was generated with MCL clustering (inflation number 5), and 8 clusters were revealed with high confidence (score >0.7) (Figure 5B), revealing the following. SHC (Src-homology 2 domain containing) Transforming protein 1 (SHC1) was the adaptor protein that interacts with DDR1 with >90% confidence. Protein Tyrosine Phosphatase non-receptor type 11 (PTPN11), a known activator of RAS signaling pathway, was another DDR1 interaction partner identified. KRAS interactors were EGFR/ERBB2 receptor tyrosine kinases, BRAF/RAF1, PIK3CA, and adaptor proteins Growth Factor Receptor Bound Protein 2 (GRB2), Son of Sevenless Homolog 1 (S0S1). Interactome for SHC1 involved GRB2, S0S1, and SRC; GRB2 connecting SHC1 with BCR, ABL1, and CTNNB1; and S0S1 connecting SHC1 with Src kinases (SRC, LYN) (Figure 5C). Interactome (with clustering) revealed BCR to interact with ABL1 (independent of gene fusions), and ABL1 to complex with CTNNB1. This was confirmed by physical and functional interactions identified between DDR1, BCR and ABL1 based on Pathway Commons database (Figure 5C). Together this indicates signaling relay from DDR1 (as homodimer or heterodimer with EGFR/ERBB2) via SHC1, GRB2, S0S1 to BCR and, from BCR-ABL 1-CTNNBl complex to KRAS.

Combinatorial efficacy ofNilotinib with Lapatinib

Because nilotinib exhibited a higher inhibitory effect as single agent, and equivalent percent inhibition to other BCR-ABL MTKs, when administered in combination with Lapatinib, its efficacy was evaluated in clonal cell proliferation assays. Comparable cytotoxic effects of Nilotinib and Bafetinib were observed when combined with Lapatinib in clonal proliferation of HCT116, COAD line harboring KRASG13D mutation (Figure 6B), and Nilotinib in combination was also effective against proliferation of KRASWT COAD line, CaCo2 (Figure 18B). Nilotinib plus Lapatinib administration impacted multi- spheroid viability for both KRAS-mutant COAD lines, HCT116 and SW480, (Figure 6 A, 6C) and their synergy was confirmed in HCT116 multispheroids (Figure 16A-16D). Since nilotinib and lapatinib are brain penetrant, investigating whether their combination can offer better outcome in GBM tumoroids cultured in vitro revealed loss of spheroid viability in GBM line U251. The two drugs synergized in U251 multi- spheroids (Figures 6E, Figure 16E-16H). DDR1 is a common target of all BCR-ABL multi-tyrosine kinase inhibitors (Holland et al., Nat. Genet., 25(1) : 55-7 (2000)). The effect of DDR1 specific inhibitor, DDR1 7RH, was also evaluated on clonal cell proliferation of HCT116, and on spheroid viability of GBM (U251) line and observed significantly enhanced cytotoxicity in combination with lapatinib (Figure 18C-18D, Figure 6D).

Targeting DDR1/BCR-ABL1 with EGFR/ERBB2 against radio-resistance

The cytotoxic potential of the combination of drugs Bafetinib and Nilotinib with or without Lapatinib, was evaluated by administering the drugs on irradiated multi- spheroids from HCT116 and measuring spheroid viability and Caspase 3/7 activity (apoptosis) at Day 5 post-IR. Significant reduction in spheroid viability and increase in apoptosis were observed in combination treatments (Bafetinib or Nilotinib plus Lapatinib) as compared to control groups at all IR doses administered (Figure 7A). The combinatorial effect of Nilotinib plus lapatinib was further confirmed on patient derived GBM (PD-GBM) lines, GBM965 and QNS108 (intrinsically radioresistant), and both the lines showed pronounced growth reduction in combination treatment (Figure 7B and 7C). To evaluate the effect of targeting DDR1/BCR- ABL1 axis in combination with Lapatinib on multi-spheroid viability, the PD-GBMs were cultured for 6 days, drugs were administered, and viability was measured at day 2-post treatment. Significant reduction in viability was observed with for all three combinations (Nilotinib, DDR1 7RH or GMB475) with Lapatinib. Fold change between drug alone and drug+Lapatinib was comparable in both the lines for Nilotinib and was relatively higher for DDR1 7RH and GMB475 in QNS108. Relatively high percent inhibition values were obtained for GBM965 when administered with either of the three drugs as single agent or in combination, which could be due to cell line specific intrinsic genomic or proteomic profiles that account for its survival and growth. To evaluate this, the Gene expression dataset (GSE144610) available for GBM965 was used (Figure 19). Significantly higher reads (counts-normalized) were observed for genes EGFR, ERBB2 as compared to ERBB3/4, high expression of BCR, ABL1, KRAS, BRAF, and CTNNB1 (P-catenin). Positive regulators of KRAS signaling (PTPN2 and PTPN11) had higher read counts than NF 1 gene (negative regulator of KRAS). Moreover, PI3K/MTOR proteins had higher read counts as opposed to their negative regulator protein PTEN. Being a proliferative line, and, with high intrinsic radioresistance, it is likely to engage activated WNT and KRAS crosstalk, which led to its observed sensitivity to combinatorial targeting of DDR1/BCR-ABL and EGFR/ERBB2. The results provided herein demonstrate that five inhibitors (PLX8394, Bafetinib, Ponatinib, VU6015929, and DDR1 7rh) can each be used in combination with one or more EGFR inhibitors (e.g., Lapatinib or Afatinib) to treat cancer. As demonstrated herein, Bafetinib and Ponatinib exhibited substantial responses in all COAD lines, independent of their KRAS- mutation type and status (Figures 1 A-1D, 2A-2C, and 3A-3D). The overlapping targets among Bafetinib, Ponatinib, VU6015929, and DDR1 7rh included (i) DDR1 protein, a receptor tyrosine kinase involved in cell-cell and cell-matrix adhesion and upregulated in several cancers. DDR1 was identified as a common target among all 4 drugs (Bafetinib, Ponatinib, DDR1 7rh, and VU6015929); (ii) Src kinases (Lyn/Src); (iii) break-point cluster (BCR) protein (an activator of GEF (Guanosine nucleotide exchange factor), involved in oncogenic transformations)); and (iv) ABL/ABL1 kinase (involved in growth, survival and cytoskeletal remodeling). BCR-ABL fusion is a genomic aberration in chronic myelogenous leukemia (CML); and, independent of this genomic alteration, BCR and ABL proteins cooperate to activate KRAS signaling (Ren, Nat Rev Cancer 5: 172-183 (2005); and Ting etal., FASEB J. 29(9):3750-3761 (2015)). DDR1 was shown as a therapeutic target in colon cancer, having BCR signaling downstream (Jeitany et al.. EMBO Mol Med. 10(4):e7918 (2018)). SRC kinases are known to associate with receptor tyrosine kinases (including EGFR, DDR1), and regulate BCR activation (Leitinger, IntRev Cell Mol Biol 310:39-87 (2014); Majo and Auguste, Cancers (Basel) \3(Ty. Y125 (2021); and Meyn et al., J Biol Chem. 281(41):30907-30916 (2006)); together this explains an active signaling network, involving KRAS, SRC, DDR1, BCR, and ABL proteins.

A synergy of these five drugs was observed with both Lapatinib and Afatinib, with best scores obtained for Lapatinib in COAD (Figure 4A-J, 15A-15J, and Table 9). Table 9 provides the cumulative synergy- and antagonism- scores obtained for each of the drugs, the doses of Drug- 1 and Drug-2 at which synergy was maximum, and the maximum synergy score value for each of the drug combination tested.

Table 9. Synergy Scores for Lapatinib and Nilotinib in HCT116 and U251.

DDR1 was originally identified as a neurotrophic receptor tyrosine kinase (NTRK4), highly upregulated in brain cancers (Vehlow et al., Cell Rep. 26(13):3672-3683 (2019)), and a significant alteration frequency (46%) in EGFR gene is observed in brain tumors. Therefore a comparative investigation of DDR1 targeting in combination with an EGFR inhibitor (Lapatinib) was conducted in an additional tumor model of GBM, and the brain penetrable DDR1/BCR-ABL inhibitor Nilotinib was included to evaluate its combinatorial efficacy. The data showed significant cytotoxicity by Nilotinib plus Lapatinib treatment in both COAD and GBM lines, and, in cells that recovered from radiation-stress induced by 20Gy-IR (Figures 4A-4F, 5A-5E, and 6A-6E). At the genomic level, transcriptional expression of genes EGFR, IGF1R, BCR, ABL and APC positively correlated with DDR1 in COAD patient datasets, and the expression of genes for EGFR, BCR, and sternness (SOX2, SOX9) correlated with expression of DDR1 and KRAS in PanCancer. The protein interactome of DDR1 with adaptor proteins (SHC1, GRB2, and S0S1) was investigated to understand how these connect DDR1 to SRC, BCR, ABL, KRAS, and Phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) signaling. This analysis revealed BCR-ABL mediated P-catenin activation downstream of DDR1/SRC signaling, and KRAS- activator SHP2 (PTPN11) being one of the interaction partners of DDR1, indicating implications of DDR1 in cancer stem cell activation and proliferation (Figures 5A-5H). Taken together, these results demonstrate that targeting DDR1-, BCR-ABL- or DDR1/BCR-ABL with EGFR/ERBB2 could offer a potential therapeutic strategy against WNT activated (stem-like), and KRAS mutant colorectal adenocarcinoma, with translatable applications in glioblastoma.

These results demonstrate the identification of the signaling pathways that could be targeted in combination with small molecule inhibitors of Wild-type EGFR to overcome the stem-like KRAS mutant phenotype in tumoroids of colorectal adenocarcinoma (COAD). These involved three combinatorial strategies (i) Targeting DDR1, BCR-ABL and EGFR-ERBB2/4 (ii) Targeting Pan-RAF/BRAF with EGFR-ERBB2/4 and (iii) Targeting PI3K, MTOR and EGFR- ERBB2/4 (schemes illustrated in Figure 7D). Among these three approaches, targeting of DDR1/BCR-ABL with EGFR-ERBB2/4 was an emphasis and was identified it to be effective independent of KRAS-mutation type and status in COAD. Moreover, its significance was validated in additional highly treatment resistant tumor model of Glioblastoma (GBM), confirming the efficacy of our prime combination in patient derived primary GBMs. These results also demonstrate a comprehensive investigation on identifying targeted therapies against Cetuximab resistance stem-like KRAS-driven tumors, by exploring the inhibitors against a wide spectrum of signaling pathways and, revealing the complementation of DDR1/BCR/ABL axis with EGFR-ERBB2/4, which could be harnessed to combat chemoradioresistance mediated by high-P-catenin and activated-KRAS. Underpinning the mechanisms of how DDR1/BCR-ABL targeting works against KRAS-mutant tumors requires further investigation, however, it may involve limiting the stimuli for cell adhesion, receptor trafficking, survival, anti-apoptotic and proliferation factors including signaling adaptors and phosphatases, all of which may co-operate with mutant-KRAS to exhibit tumor aggressivity. The results provided herein demonstrate an unprecedented role of the interactome of DDR1, BCR and ABL1 proteins with EGFR-ERBB2-4 signaling in tumor progression and poor patient outcome. While the only FDA approved drugs currently available for DDR1 and BCR/ABL proteins are the BCR-ABL family of multi-tyrosine kinase inhibitors, however, DDR1- and BCR-ABL-specific compounds are constantly being developed (Canning et al., J. Mol. Biol., 426(13):2457-70 (2014); and Elkamhawy et al., Int. J. Mol. Sci., 22(12): 6535 (2021)), and a combination of DDR1-, BCR-ABL1-, and EGFR- ERBB2/4 specific drugs could cooperate and synergize and open a new paradigm for future cancer therapeutics.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.