Technical field

The invention relates to compositions for use in a method of treatment of B cell acute lymphoblastic leukemia (B-ALL), diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), or T-cell acute lymphoblastic leukemia (T-ALL), the compositions comprising at least one Menin inhibitor and optionally a Class I HDAC inhibitor. The invention further relates to a kit of parts comprising at least two recipients, wherein one recipient comprises at least one Menin inhibitor and the other recipient comprises at least one Class I HDAC inhibitor. The invention further relates to the compositions as described herein for use in a method of inducing expression of HDAC7 in malignant hematologic cells.

Background of the invention

Acute lymphoblastic leukemia derived from B cell progenitors (pro-B cells) (pro-B-ALL) is the most prevalent type of malignancy among the 350 new infants that are annually diagnosed of leukemia in Spain. Despite the fact that 80% of B-ALL remission has been achieved due to recent therapeutic advances, a specific subgroup of patients that present the t(4;11) rearrangement commonly relapse, converting acute leukemia into the main cause of pediatric cancer-associated death. Current therapy in this group of patients consists of an initial phase that includes prednisone and L-asparaginase, followed by an induction stage with cytarabine, vincristine and daunorubicin, since it is believed that leukemic cells may derive from a myeloid progenitor. However, survival rate of infants (< 1 year old) diagnosed with t(4; 11)-rearranged pro-B-ALL does not even reach 35%. In fact, due to their young age and vulnerability, these infants are normally excluded from clinical trials. There is, thus, an urgent need of improving current therapy and implementing novel combinatorial treatments and precision medicine.

The generation of properly differentiated hematopoietic cell populations is one of the major focuses of research on leukemia. In this area, the function of key lineage-specific transcription factors that promote cell differentiation processes is well established. However, increasing evidence demonstrates that lymphocyte-specific factors not only induce the expression of B-cell specific genes, but they also lead to the repression of lineage- inappropriate genes ensuring the correct identity of B lymphocytes. Therefore, the aberrant expression and/or mutation of many of these factors involved in B lymphocyte development have been linked to the outcome of hematopoietic malignancies, such as pro-B-ALL. For instance, overexpression of the transcription factor c-MYC is known to be involved in B-ALL and other type of hematological malignancies derived from B lymphocytes.

During terminal differentiation in peripheral lymphoid organs, B cells generate a hugely diverse repertoire of antibodies, which enable specific immune responses against pathogens that the organism may encounter. The generation of the antibodies by mature B cells after facing the antigen takes place in the germinal centers (GCs). As it occurs during earlier stages of B cell development, an aberrant regulation of key transcription factors at this point also triggers the onset of hematological malignancies, such as diffuse large B cell lymphoma (DLBCL) or follicular lymphoma (FL). Both DLBCL and FL are frequent types of non-Hodgkin lymphoma (NHL). On the one hand, FL is an indolent and slow-growing form of lymphoma, while DLBCL is the most aggressive type of NHL worldwide, accounting for a total of 40% of all NHL. DLBCL presents large genetic and clinical heterogeneity and a significant subset relapse after regular chemotherapeutic treatment, which associates with decreased survival.

Histone or protein deacetylases (HDACs) are epigenetic regulators that have emerged as crucial transcriptional repressors in highly diverse physiological and pathological systems. To date, 18 human HDACs have been identified and grouped into four classes: Class I HDACs (HDAC1, 2, 3, and 8), class II HDACs (HDAC4, 5, 6, 7, 9, and 10), class III HDACs, also called sirtuins (SIRT1, 2, 3, 4, 5, 6, and 7), and class IV HDAC (HDAC11). Class II HDACs are further subdivided into class Ila (HDAC4, 5, 7, 9) and class lib (HDAC6 and 10) groups. The emergence of HDACs as regulators at diverse steps of immune system differentiation has set them as potential candidates of therapeutic strategies aiming to treat childhood leukemias. Mutation and/or abnormal expression of class Ila HDACs have been frequently observed in disease, particularly in cancer, making them important therapeutic targets. Consequently, histone deacetylase inhibitors (HDIs) have become promising therapeutic agents.

However, we are far from fully understanding the contribution of individual HDACs to cancer. In fact, some HDACs may be under expressed or present inactivating mutations. Up to date, the approval of HDAC inhibitors has been restricted to pan-HDAC inhibitors for the treatment of T-cell malignancies and multiple myeloma. However, given the potential function of HDAC members like HDAC7 in pro-B-ALL, a global repression of all HDAC subclasses is potentially detrimental for patients. In this sense, and in an attempt to avoid the harmful effects of inhibition of all HDACs, new drugs with selective HDAC subclass inhibition are being developed. In this sense, subclass I inhibitor Chidamide, has been already tested in hematological malignancies that are not derived from B lymphocytes, such as myeloid leukemia or acute myeloid leukemia (AML), but harbor MLL rearrangements (Ye et al. Clinical Epigenetics

2019)., One of these MLL rearrangements is a t(4;11) rearrangement resulting in expression of a MLL-AF4 fusion protein that activates several genes associated to leukemogenesis through a process that involves a variety of cofactors. Class I HDACs, like HDAC1 and HDAC2, are involved in this transcriptional machinery and, in consequence, targeting these two class I HDACs with specific inhibitors like Chidamide can reduce these hematological cell malignancy..

The histone deacetylase HDAC7 has been identified as a novel biomarker that determines prognosis in t(4;11) pro-B-ALL patients (de Barrios O et al. Leukemia, 2021). De Barrios et al. provide data that strongly supports the use of HDAC7 expression levels as a prognostic marker in infant pro-B-ALL with t(4;11) rearrangement. Data has been obtained in primary samples from pro-B-ALL diagnosed infants and cell lines with t(4;11) translocation, such as SEM-K2. This cell line is derived from peripheral blood of a 5-year-old female at relapse. HDAC7 expression was significantly decreased in this cell line, as well as in infants with t(4;11) pro-B-ALL, when compared to samples from pediatric patients with an alternative rearrangement, such as t(9;11), or an unaltered KMT2A gene, which encodes the MLL protein (Pieters R et al. Lancet, 2007; 370:240-50).

When combining data from all subtypes of leukemia, patients displaying low or intermediate levels of HDAC7 present the poorest survival. In consequence, the absence of HDAC7 seems to be a key element in determining the poor outcome associated to t(4; 11 ) infant pro- B-ALL.

It has also been shown that HDAC7 is not only under expressed in pro-B-ALL cells, but that these cells need to maintain HDAC7 at very low levels of expression, in order to maintain an aberrantly active proliferation rate (de Barrios O et al. Leukemia, 2021). In this publication the authors also found that key cancer hallmarks such as cellular differentiation and apoptosis were altered. For instance, the pro-apoptotic marker MMP9 (Yadav et al. Cell Death Dis

2020) and the marker of proper B cell differentiation CD86 (Engel et al. Blood 1994) were triggered by the induction of HDAC7. Oppositely, asparagine synthetase (ASNS) was downregulated in SEM-K2 cells expressing HDAC7. This gene is associated to a worse prognosis in pro-B-ALL, since it blocks the early response to state-of-the-art treatment with L- asparaginase. The authors also found that t(4;11) infants with the lowest expression of HDAC7 displayed significantly increased levels of the chemoresistance marker ASNS, indicating that lack of HDAC7 implies an increased resistance to standard treatment.

Therefore, it can be concluded that a forced induction of HDAC7 in this highly aggressive subtype of pro-B-ALL prevents the uncontrolled proliferation of malignant cells and confers them a higher sensitivity to current chemotherapy.

The current working hypothesis is therefore that the restoration of HDAC7 expression constitutes a promising therapeutic strategy in terms of improving survival among t(4; 11) pro- B-ALL patients since it reduces the leukemogenic capacity of the cells.

It was, therefore, a target of present invention to identify a method of increasing the expression of HDAC7 in malignant hematologic cells and to provide pharmaceutical compositions that can be administered to patients suffering from pro-B-ALL that are capable of increasing the expression of HDAC7. It was a further goal of present invention to identify a combinatorial precision therapy that in addition to inducing HDAC7 expression, can enhance its leukemia suppressor function.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 : Use of Menin inhibitor MI-538 and class I HDAC selective inhibitor Chidamide to trigger HDAC7 expression in t(4;11) pro-B-ALL cell lines. (A) Protein levels of HDAC7 in SEM-K2 and RS4;11 cells. (B) gRT-PCR for HDAC7 in SEM-K2 cells treated with MI-538 (1 pM, 6 days) Chidamide (1 pM, 48 hours), or DMSO as control. (C) Relative protein expression of HDAC7 in SEM-K2 and RS4;11 cells.

Figure 2 Use of MI-538 and Chidamide in combination exerts a surprising effect in reducing t(4;11) pro-B-ALL cells viability. (A) Protein levels of HDAC7 in SEM-K2 and RS4;11 cells

(t(4;11) translocation); and REH cell line (no chromosomal alteration at KMT2A gene), - actin was used as loading control. (B) MTT assay to assess cell viability in SEM-K2, RS4;11 and REH cell lines treated with MI-538 (1 pM, 6 days) and/or Chidamide (1 pM, 48 hours), or DMSO as control.

Figure 3 Synergistic induction of HDAC7 after combinatorial therapy with MI-538 and

Chidamide alters the expression of key HDAC7 targets. gRT-PCR was performed for MMP9 (involved in apoptosis), CD86 (marker of proper B lymphocytes differentiation) and ASNS (chemoresistance marker in infant leukemia) in SEM-K2 cells treated with MI-538 (1 pM, 6 days) and/or Chidamide (1 pM, 48 hours), or DMSO as control. Figure 4 Effect of HDAC7 expression on the regular and standard therapy that is commonly used in clinical settings in infant t(4;11) pro-B-ALL patients. SEM-TetOn-Tight-HDAC7 cell line was induced with doxycycline. As shown in Figure 4, HDAC7 induction endows SEM-K2 cells with a higher sensitivity to prednisone treatment, since the reduction of viability is significant at a 10-time lower dose than in normal conditions (i.e. with low HDAC7 levels).

Figure 5 MTT assay to assess cell viability in SEM-K2 and REH cell lines treated with MI-

503 (1 pM, 6 days) and/or Chidamide (1 pM, 48 hours), or DMSO as control.

Figure 6 MTT assay to assess cell viability in SEM-K2 and REH cell lines treated with MI-

463 (1 pM, 6 days) and/or Chidamide (1 pM, 48 hours), or DMSO as control.

Figure 7 MTT assay to assess cell viability in SEM-K2 and REH cell lines treated with MI-

3454 (1 pM, 6 days) and/or Chidamide (1 pM, 48 hours), or DMSO as control.

Figure 8 MTT assay to assess cell viability in RS4;11 cell lines treated with MI-503, MI-463 or MI-3454 (1 pM, 6 days) and/or Chidamide (1 pM, 48 hours), or DMSO as control.

Figure 9 gRT-PCR for HDAC7 in RS4;11 cells treated with MI-503, MI-463 or MI-3454 (1 pM,

6 days) and/or Chidamide (1 pM, 48 hours).

Figure 10 Protein levels of HDAC7 in SEM-K2 cells treated with MI-503 or MI-463 (1 pM, 6 days) and/or Chidamide (1 pM, 48 hours), or DMSO as control.

Figure 11 Schematic representation of the experimental procedure employed to validate inclusion of MI-538 to conventional chemotherapy regimen (VxL). The experimental groups are specified in the top right corner.

Figure 12 Schematic representation of the experimental procedure employed to validate

HDAC7-inducing combined therapy consisting of MI-538 and Chidamide, compared to conventional chemotherapy regimen (VxL). The experimental groups planned are specified on top right corner.

Figure 13 Addition of MI-538 improves the efficiency of conventional chemotherapy in t(4; 11) pro-B-ALL - (A) Percentage of t(4;11) pro-B-ALL cells detected in bone marrow aspirates before (Day 0) and after (Day 21) treatment. Left panel corresponds to control untreated mice. Mice treated with VxL are shown in central panel, while mice treated with VxL + MI-538 are shown in right panel. Dashed grey line indicates the average of all mice in each group. Dotted line is placed at 1% of pro-B-ALL cells engraftment level, indicating threshold for complete remission. (B) Comparison of t(4;11) pro-B-ALL cells engraftment before the treatment (Day 0), demonstrating no significant differences between both experimental groups. (C) Comparison of t(4; 11) pro-B-ALL cells engraftment after the treatment (Day 21), showing a significant reduction of engraftment in mice treated with MI-538.

Figure 14: Addition of MI-538 reduces the risk of recurrence after treatment with conventional therapy - (A) Percentage of t(4;11) pro-B-ALL cells detected in peripheral blood aspirates before (Day 0) and after (Days 15, 28 and 42) treatment. Left panel corresponds to control untreated mice. Mice treated with VxL are shown in central panel, while mice treated with VxL + MI-538 are shown in right panel. Dashed grey line indicates the average of all mice in each group. Dotted line is placed at 10% of pro-B-ALL cells engraftment level, indicating threshold for recurrence of the disease. (B) Comparison of t(4;11) pro-B-ALL cells engraftment in peripheral blood (PB) at Day 42 after treatment initiation. A clear tendency towards a reduction of engraftment in mice treated with VxL + MI-538 was observed.

Figure 15: Addition of MI-538 reduces engraftment of t(4; 11) pro-B-ALL cells five weeks after treatment completion - (A) Percentage of t(4;11) pro-B-ALL cells detected in bone marrow aspirates before (Day 0) and after (Days 21 and 50) treatment. Left panel corresponds to control untreated mice. Mice treated with VxL are shown in central panel, while mice treated with VxL + MI-538 are shown in right panel. Dashed grey line indicates the average of all mice in each group. (B) Comparison of t(4;11) pro-B-ALL cells engraftment before the treatment (Day 0), demonstrating no significant differences between both experimental groups. (C) Comparison of t(4;11) pro-B-ALL cells engraftment five weeks after treatment completion (Day 50), showing a significant reduction of engraftment in mice treated with VxL and MI-538, compared to those treated with VxL alone.

Figure 16: Addition of MI-538 reduces engraftment of t(4; 11) pro-B-ALL cells five weeks after treatment completion - (A) Percentage of t(4; 11) pro-B-ALL cells detected in peripheral blood aspirates before (Day 0) and after (Days 15, 28, 42 and 50) treatment. Left panel corresponds to control untreated mice. Mice treated with VxL are shown in central panel, while mice treated with VxL + MI-538 are shown in right panel. Dashed grey line indicates the average of all mice in each group. (B) Comparison of t(4; 11) pro-B-ALL cells engraftment in peripheral blood (PB) at Day 50 after treatment initiation. A clear tendency towards a reduction of engraftment in mice treated with VxL + MI-538 is observed, despite differences are not significant.

Figure 17: Addition of MI-538 reduces engraftment of t(4;11) pro-B-ALL cells in spleen five weeks after treatment completion - (A) Weight of spleen at Day 50 after treatment initiation. A clear tendency towards a reduction of spleen weight in mice treated with VxL + MI-538 is observed, despite differences are not significant. (B) As in (A), but calculating the percentage of spleen weight compared to initial mouse body weight. (C) Picture of spleens from panels (A) and (B), compared to control untreated spleen. (D) Percentage of t(4;11) pro-B-ALL cells detected in spleen at Day 50 after treatment initiation, showing a significant reduction of engraftment in mice treated with VxL and MI-538, compared to those treated with VxL.

Figure 18: (A) RNA seguencing analysis of HDAC7 normalized counts of 63 diffuse large B cell lymphoma (DLBCL) versus 4 healthy Germinal Centre (GC B cells) control samples. (B) Relative HDAC7 mRNA guantification of GC B cells from tonsils (n=4), and DLBCL patients. ABC and GCB refer to two distinct subtypes of DLBCL (n=3, per lymphoma subtype). (C) Kaplan-Meier plot for overall survival of 292 DLBCL patients based on HDAC7 expression. Grey line corresponds to patients with high HDAC7 expression and black line corresponds to patients with low HDAC7 expression. Patients are split in groups by median expression of HDAC7. Data is represented as mean ± Standard Error (SE) of above-mentioned independent experiments. * p<0.05; ** p<0.01 ; *** p<0.001; unpaired Student’s t test.

Figure 19: (A) MD901 cells were transduced to express HDAC7 in a doxycycline (D) inducible manner (Tet-On-Tet-Tight-HDAC7), and treated, or not, for 72 hours. At the indicated times of treatment with doxycycline, cell number was determined by cell counting (n=3 per condition). (B) Absorbance units were measured in MTT assay, indicating cell viability. E (Tet-On-Tet-Tight-Empty vector), H7 (Tet-On-Tet-Tight-HDAC7) (n=3 per condition. (C) Representative scheme of the xenografts assay to assess whether HDAC7 alters tumor growth in DLBCL MD901 cells in vivo. (D) Image of the extracted tumors from (C) at day 11 post-treatment (n=11 per condition). (E) Relative volume guantification of tumors shown in (D). (F) Weight average of tumors extracted in (D) at day 11 post-treatment. Data is represented as mean ± SE of above-mentioned independent experiments. * p<0.05; ** p<0.01; *** p<0.001 ; unpaired Student’s t test. SUMMARY OF THE INVENTION

In one aspect the present invention relates to a composition, preferably a pharmaceutical composition, for use in a method of treatment of B cell acute lymphoblastic leukemia (B- ALL), diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL) or T-cell acute lymphoblastic leukemia (T-ALL), preferably B cell acute lymphoblastic leukemia (B-ALL), wherein the composition comprises at least one Menin inhibitor.

In one embodiment of the present invention said composition is administered to a subject in need thereof and said administration induces expression of HDAC7 in malignant hematologic cells.

In a further embodiment said B cell acute lymphoblastic leukemia (B-ALL) is characterized in that the B-ALL cells comprise a t(4; 11) rearrangement.

In a further aspect the present invention relates to a composition, preferably a pharmaceutical composition, for use in a method of inducing expression of HDAC7 in malignant hematologic cells, wherein the composition comprises at least one Menin inhibitor.

In one embodiment of present invention the at least one Menin inhibitor is a compound according to formula (A) wherein

Ri is a C1-4 alkyl group including branched alkyl optionally substituted with up to three halogen atoms;

R2 is H or NRaRb, Ra and Rb are independently H or C1-4 alkyl;

Ra is H or OH; R4 independently represents H, C1-4 alkyl group including cycloalkyl, or aryl group including heteroaryl.

R4 is optionally substituted with Re, where Re represents H, NRcRd and Rc and Rd are independently H, C1-4 alkyl group including branched alkyl optionally substituted with up to three halogen atoms, CHO or CONRcRd n is 0 - 4

Rs is H or a C1-4 alkyl group including branched alkyl optionally substituted with up to three halogen atoms.

In a further aspect the at least one Menin inhibitor is a compound according to formula (A), wherein

Ri is -CH2CF3

R ₂ is H or MeNH-

R3 is H or OH

R4 independently represents H, a 4-pyrazolyl group (I) or a substituted bicyclic alkyl group n is 0 - 4

Rs is H or methyl.

In a preferred aspect the at least one Menin inhibitor is a compound according to formula (A), wherein

R1 is -CH2CF3

R ₂ is H

R3 is H or OH

R4 is H or a 4-pyrazolyl group n is 0 or 1

Rs is H or methyl. In an even more preferred aspect, the at least one Menin inhibitor is a compound according to formula (A), wherein

Ri is -CH2CF3

R2 and Rs are H

R ₃ is OH

R4 is a 4-pyrazolyl group - n is 1.

In one embodiment of the present invention the Menin inhibitor is selected from the group consisting of MI-538, MI-503, MI-463, MI-3454, MI-1481 or MI-2, or a combination thereof. Preferably the Menin inhibitor of present invention is selected from MI-538, MI-503, MI-463 or MI-3454, most preferred is MI-538.

In a further embodiment of present invention, the expression of HDAC7 is mRNA expression and said mRNA expression of HDAC7 is increased by at least about 1.2-fold, 1.3-fold, 1.4- fold, 1.5-fold in the malignant hematologic cells compared to the mRNA expression of HDAC7 in reference cells.

In yet a further embodiment of present invention the expression of HDAC7 is protein expression and said protein expression of HDAC7 is increased by at least about 1.5-fold, 2.0-fold, 2.5-fold, 3.0-fold, 3.5-fold, 4.0-fold, 4.5-fold, 5.0-fold, 5.5-fold, 6.0-fold, 6.5-fold, 7.0- fold, 7.5-fold, 8.0-fold, 8.5-fold, 9.0-fold, 9.5-fold, 10.0-fold, 10.5-fold, 11.0-fold, 11.5-fold, 12.0-fold in the malignant hematologic cells compared to the protein expression of HDAC7 in reference cells.

In one embodiment of present invention the malignant hematologic cells are selected from B cell acute lymphoblastic leukemia (B-ALL) cells, diffuse large B cell lymphoma (DLBCL) cells, follicular lymphoma (FL) and T-cell acute lymphoblastic leukemia (T-ALL) cells. In a preferred embodiment the B-ALL cells are B cell acute lymphoblastic leukemia (B-ALL) cells, in the most preferred embodiment the B-ALL cells pro-B-ALL cells characterized by a t(4;11) rearrangement. In a further embodiment of present invention, the composition further comprises at least one Class I HDAC inhibitor. The at least one Class I HDAC inhibitor can be selected from the group consisting of Chidamide, Entinostat, Moceti nostat, Tacedinaline or Romidepsin, or a combination thereof. A preferred Class I HDAC inhibitor of present invention is Chidamide.

In one embodiment of present invention the at least one Menin inhibitor is MI-538 and the at least one Class I HDAC inhibitor is Chidamide.

The present invention further relates to a kit of parts comprising at least two recipients, wherein one recipient comprises at least one Menin inhibitor and the other recipient comprises at least one Class I HDAC inhibitor.

In one embodiment the kit of parts comprises at least two recipients, wherein one recipient comprises at least one Menin inhibitor as described herein, preferably said Menin inhibitor is selected from the group consisting of MI-538, MI-503, MI-463, MI-3454, MI-1481 or MI-2, or a combination thereof, more preferred the group consisting of MI-538, MI-503, MI-463 and MI-3454, most preferred the Menin inhibitor being MI-538, and the other recipient comprises at least one Class I HDAC inhibitor selected from the group consisting of Chidamide, Entinostat, Mocetinostat, Tacedinaline or Romidepsin, preferably wherein the at least one Class I HDAC inhibitor is Chidamide.

In one preferred embodiment of present invention the kit of parts comprises at least two recipients, wherein one recipient comprises MI-538 and the other recipient comprises Chidamide.

The present invention furthermore relates to the composition as disclosed herein above for use in a method of treatment of B cell acute lymphoblastic leukemia (B-ALL), diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL) or T-cell acute lymphoblastic leukemia (T- ALL), preferably of B cell acute lymphoblastic leukemia (B-ALL).

The present invention furthermore relates to a method for treating B cell acute lymphoblastic leukemia (B-ALL), diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL) or T-cell acute lymphoblastic leukemia (T-ALL), preferably B cell acute lymphoblastic leukemia (B- ALL) in a subject in need thereof, comprising administering to said subject the composition as defined herein above. DETAILED DESCRIPTION OF THE INVENTION

The present invention may be understood more readily by reference to the following detailed description of the preferred embodiments of the invention, and to the examples included therein.

The restoration of HDAC7 expression constitutes a promising therapeutic strategy in terms of improving survival among t(4;11) pro-B-ALL patients since it reduces the leukemogenic capacity of the cells. It was therefore a target of present invention to identify a method of increasing the expression of HDAC7 in malignant hematologic cells and to provide pharmaceutical compositions that can be administered to patients suffering from pro-B-ALL that are capable of increasing the expression of HDAC7. It was a further goal of present invention to identify a combinatorial precision therapy that, in addition to inducing HDAC7 expression, can enhance its leukemia suppressor function.

The present invention therefore relates to a composition, preferably a pharmaceutical composition, for use in a method of inducing expression of HDAC7 in malignant hematologic cells, wherein the composition comprises at least one Menin inhibitor.

Menin inhibitors are novel targeted agents currently in clinical development for the treatment of genetically defined subsets of acute leukemia. Menin has a tumor suppressor function in endocrine glands. Germline mutations in the gene encoding Menin cause the multiple endocrine neoplasia type 1 (MEN1) syndrome, a hereditary condition associated with tumors of the endocrine glands. However, Menin is also critical for leukemogenesis in subsets driven by rearrangement of the Lysine Methyltransferase 2A (KMT2A) gene, also known as mixed lineage leukemia (MLL), which encodes an epigenetic modifier.

Whilst Menin inhibitors and their various uses have been known for some time, their effect on the expression of HDAC7 in malignant hematologic cells has not been previously described. The inventors have now surprisingly found that Menin inhibitors can induce the expression of HDAC7 in such cancer cells, thus providing a potential new treatment of diseases caused by reduced cellular HDAC7 expression, such as B cell acute lymphoblastic leukemia (B-ALL), diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL) or T-cell acute lymphoblastic leukemia (T-ALL).

The inventors have used SEM-K2 and RS4;11 cell lines harboring t(4;11) translocation and have shown that when incubated with the Menin inhibitor MI-538, a clear upregulation of HDAC7 at protein levels in both cell lines (Figure 1A) and also at mRNA level in SEM-K2 cells (Figure 1B) could be observed (see Example 1). In order to enhance the effect of the Menin inhibitor the inventors added the class I HDAC selective inhibitor Chidamide. As can be seen from Figure 1 B, in SEM-K2 cells, treatment with Chidamide alone does not provide any increase in mRNA expression of HDAC7, whereas treatment with MI-538 alone leads to an about 1.5-fold increase of mRNA expression of HDAC7 in the cells. Surprisingly, the combination of Chidamide and Menin inhibitor MI-538 provides a significant and synergistic increase in comparison to the use of Chidamide or MI-538 alone in SEM-K2 cells.

The increase of the mRNA expression of HDAC7 of at least 1.2-fold will already show an effect on the cell viability. The increase of about 1.5-fold as for Menin inhibitor MI-538 is therefore striking and leads to the strong effects on the cell viability when MI-538 is used alone and/or in combination with Chidamide as shown in Fig.2B.

In a further embodiment of present invention, in which the expression of HDAC7 refers to the mRNA expression, said mRNA expression of HDAC7 is increased by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold in the malignant hematologic cells compared to the mRNA expression of HDAC7 in reference cells. It is to be understood that the reference cells refer to the same type of malignant hematologic cells as the ones being assessed for their increase in mRNA expression of HDAC7, but which have not been treated with the composition as disclosed herein.

As could be shown in the cell viability assays performed by the inventors and as shown in the Examples, such an increase of HDAC7 mRNA expression leads to decreased cell viability in the malignant hematologic cells.

In yet a further embodiment of present invention the expression of HDAC7 is protein expression and said protein expression of HDAC7 is increased by at least about 1.5-fold, 2.0-fold, 2.5-fold, 3.0-fold, 3.5-fold, 4.0-fold, 4.5-fold, 5.0-fold, 5.5-fold, 6.0-fold, 6.5-fold, 7.0- fold, 7.5-fold, 8.0-fold, 8.5-fold, 9.0-fold, 9.5-fold, 10.0-fold, 10.5-fold, 11.0-fold, 11.5-fold, 12.0-fold in reference cells. It is to be understood that the reference cells refer to the same type of malignant hematologic cells as the ones being assessed for their increase in protein expression of HDAC7, but which have not been treated with the composition as disclosed herein.

In one embodiment of present invention the malignant hematologic cells are selected from B cell acute lymphoblastic leukemia (B-ALL) cells, diffuse large B cell lymphoma (DLBCL) cells, follicular lymphoma (FL) and T-cell acute lymphoblastic leukemia (T-ALL) cells. In a preferred embodiment the B-ALL cells are B cell acute lymphoblastic leukemia (B-ALL) cells, in the most preferred embodiment the B-ALL cells pro-B-ALL cells characterized by a t(4;11) rearrangement.

MTT viability assays including previously used SEM-K2 and RS4;11 cells, together with REH cells, that harbor an unaltered form of MLL gene, showed that REH cells displayed abundant levels of HDAC7, compared to t(4; 11) translocated cells (Figure 2A) and that MI-538 strongly reduced viability of both SEM-K2 and RS4;11 cells (Example 2). Strikingly, the combination with Chidamide produced an even more dramatic effect, being significantly stronger than both drugs administered alone (Figure 2B, left and center panels). The demonstration that the effect of these drugs is, at least partially, a consequence of HDAC7 induction can be extracted from the MTT assays performed in REH cell line. This cell line, with strong expression of HDAC7, did not respond to MI-538 and/or Chidamide treatments, and no effect was observed on cell viability (Figure 2B, right panel).

A forced overexpression of HDAC7 in SEM-K2 shifts the genomic profile of t(4; 11) pro-B-ALL cells towards a more anti-oncogenic profile through the altered expression of targets involved in key hallmarks of cancer, such as cell differentiation, chemotherapy resistance or apoptosis prevention. In order to corroborate whether the effect of MI-538 and Chidamide on endogenous HDAC7 can be paralleled to that of exogenous HDAC7 induction, some of HDAC7’s previously reported targets were analyzed (Example 3). It could be shown that, as expected, both MI-538 and Chidamide triggered the mRNA levels of the pro-apoptotic gene MMP9 and the marker of B lymphocyte differentiation CD86, showing a synergistic effect when both drugs were administered together (Figure 3, left and center panels). Oppositely, the chemoresistance marker ASNS is downregulated upon treatment of SEM-K2 cells with MI-538 and Chidamide (Figure 3, right panel), according to previous data showing an opposite pattern of expression with HDAC7 (de Barrios et al. 2021). Therefore, the effect of MI-538 and Chidamide not only affects HDAC7 itself, but also its previously described targets, reinforcing the potential use of this combinatorial therapy to improve outcome of t(4;11) pro-B-ALL.

As a proof-of-concept, the effect of HDAC7 expression was evaluated on the regular and standard therapy that is commonly used in clinical settings in infant t(4;11) pro-B-ALL patients. SEM-TetOn-Tight-HDAC7 cell line (also used in de Barrios et al. 2021), that permits HDAC7 induction with doxycycline treatment was used. As shown in Figure 4, HDAC7 induction endows SEM-K2 cells with a higher sensitivity to prednisone treatment, since the reduction of viability is significant at a 10-time lower dose than in normal conditions (i.e. with low HDAC7 levels).

In order to assess if this surprising effect can also be observed with other Menin inhibitors, the inventors have tested further compounds, as shown in Examples 5 to 10. As can be seen from the figures, all tested Menin inhibitors MI-3454, MI-503 and MI-463 reduce viability of SEM-K2 and RS4;11 cells. The combination with Chidamide enhanced the effect. As was expected, an upregulation of HDAC7 mRNA as well as protein expression could be shown in SEM-K2 cells, which was in line with the above findings for Menin inhibitor MI-538.

In one embodiment of present invention the Menin inhibitor is therefore a compound according to formula (A) wherein

Ri is a C1-4 alkyl group including branched alkyl optionally substituted with up to three halogen atoms;

R2 is H or NRaRb, Ra and Rb are independently H or C1-4 alkyl;

Ra is H or OH;

R4 independently represents H, C1-4 alkyl group including cycloalkyl, or aryl group including heteroaryl;

R4 is optionally substituted with Re, where Re represents H, NRcRd and Rc and Rd are independently H, C1-4 alkyl group including branched alkyl optionally substituted with up to three halogen atoms, CHO or CONRcRd; n is 0 - 4

Rs is H or C1-4 alkyl group including branched alkyl optionally substituted with up to three halogen atoms. Examples of aryl groups include phenyl and naphthyl. Examples of heteroaryl include 5 membered heteroaryl groups including pyrazole, triazole, furan, thiophene, thiazole, oxazole or 6 membered heteroaryl rings including pyridine, pyrimidine, pyrazine, pyridazine and triazine.

In a further aspect the at least one Menin inhibitor is a compound according to formula (A), wherein

R1 is -CH2CF3

R ₂ is H or MeNH-

R3 is H or OH

R4 independently represents H, a 4-pyrazolyl group (I) or a substituted bicyclic alkyl group n is 0 - 4

Rs is H or methyl.

In a preferred aspect the at least one Menin inhibitor is a compound according to formula (A), wherein

Ri is -CH2CF3

R ₂ is H

R3 is H or OH

R4 is H or a 4-pyrazolyl group n is 0 or 1

Rs is H or methyl.

In an even more preferred aspect, the at least one Menin inhibitor is a compound according to formula (A), wherein

R1 is -CH2CF3

R? and Rs are H

R ₃ is OH R4 is a 4-pyrazolyl group -

In one embodiment of the present invention the Menin inhibitor is selected from the group consisting of MI-538, MI-503, MI-463, MI-1481 , MI-3454 or MI-2, or a combination thereof. Preferably the Menin inhibitor of present invention is MI-538, MI-503, MI-463 or MI-3454, most preferred MI-538.

The inhibitors MI-538, MI-503, MI-463, MI-1481 and MI-3454 have the following structures and can all be commercially obtained under these numbers:

MI-463 MI-503 MI-3454 As shown in the Examples the addition of a class I HDAC inhibitor leads to a surprising enhancement of the effect of the Menin inhibitors. Without being bound by theory, the effect can be explained by the class I HDAC inhibitor modifying the binding of HDAC7 to its cofactors whilst the Menin inhibitor induces HDAC7. The class I HDAC inhibitor thus promotes that HDAC7 can exert its leukemia suppressor function.

In a further embodiment of present invention, the composition therefore further comprises at least one Class I HDAC inhibitor. The at least one Class I HDAC inhibitor can be selected from the group consisting of Chidamide, Entinostat, Mocetinostat, Tacedinaline or Romidepsin, or a combination thereof. A preferred Class I HDAC inhibitor of present invention is Chidamide.

In a preferred embodiment of present invention, the at least one Menin inhibitor is MI-538 and the at least one Class I HDAC inhibitor is Chidamide.

In some embodiments, the Menin inhibitor and the class I HDAC inhibitor are coadministered concurrently (e.g., simultaneously, essentially simultaneously or within the same treatment protocol) or sequentially.

In some embodiments, the Menin inhibitor and class I HDAC inhibitor are co-administered in separate dosage forms. In some embodiments, the Menin inhibitor and a class I HDAC inhibitor are co administered in combined dosage forms.

The present invention further relates to a kit of parts comprising at least two recipients, wherein one recipient comprises at least one Menin inhibitor and the other recipient comprises at least one Class I HDAC inhibitor.

In one embodiment the kit of parts comprises at least two recipients, wherein one recipient comprises at least one Menin inhibitor selected from the group consisting of MI-538, MI-2, MI-503, MI-463, MI-1481 or MI-3454, or a combination thereof, preferably wherein the Menin inhibitor is MI-538, MI-503, MI-463, or MI-3454, most preferred MI-538, and the other recipient comprises at least one Class I HDAC inhibitor selected from the group consisting of Chidamide, Entinostat or Mocetinostat, preferably wherein the at least one Class I HDAC inhibitor is Chidamide.

In one preferred embodiment of present invention the kit of parts comprises at least two recipients, wherein one recipient comprises MI-538 and the other recipient comprises Chidamide. The present invention furthermore relates to the composition as disclosed herein above for use in a method of treatment of B cell acute lymphoblastic leukemia (B-ALL).

Other malignancies related to B lymphocytes in which HDAC7 has tumor suppressor functions, apart from pro-B-ALL, are also envisaged for indications that can be treated as described herein. It has been previously shown by the inventors that in Burkitt lymphoma and adult pro-B-ALL cells display low levels of HDAC7 and, interestingly, when its expression is induced, cells lose proliferation capacity and viability (Barneda-Zahonero et al. Cell Death and Dis, 2015). For example, patients diagnosed of Diffuse Large B Cell Lymphoma or B- ALL in adults can also benefit from the treatment shown herein, see Examples 11 and 12 and figures 18 and 19. In addition, HDAC7 plays an important role in T-lymphocytes and its induction can also contribute to a better treatment in malignancies derived from T cells.

The present invention therefore furthermore relates to the composition as disclosed herein above for use in a method of treatment of B-lymphocyte related malignancies or Diffuse Large B Cell Lymphoma (DLBCL), follicular lymphoma (FL) and T-cell acute lymphoblastic leukemia (T-ALL), preferably B cell acute lymphoblastic leukemia (B-ALL).

The present invention furthermore relates to a method for treating B-lymphocyte related malignancies or Diffuse Large B Cell Lymphoma (DLBCL), follicular lymphoma (FL) and T- cell acute lymphoblastic leukemia (T-ALL), preferably B cell acute lymphoblastic leukemia (B- ALL) in a subject in need thereof, comprising administering to said subject the composition as defined herein above.

As could be shown in in vivo experiments in a leukemia murine model (see Figure 11 for the experimental design) a Menin inhibitor of present invention, specifically MI-538, was able to reduce leukemogenesis. It was demonstrated that inclusion of MI-538 together with the standard chemotherapy treatment (known as VxL) reduces the percentage of leukemic cells in bone marrow of treated mice and that there are more mice in complete remission in the MI-538 treated group when assessed after day 21 post treatment (Figure 12).

Surprisingly, the addition of MI-538 reduced the risk of recurrence after treatment with conventional therapy. As can be seen in Figure 13 at day 42 after treatment initiation recurrence is significantly lower in the MI-538 group than in the group only treated with standard chemotherapy. After 50 days post treatment a decrease of leukemic cells engraftment in bone marrow and in PB was observed. Furthermore, the weight of the spleen, which serves an additional indicator of the global engraftment of leukemic cells, was clearly lower in MI-538 treated mice.

Experiments for assessing the combination therapy of a Menin inhibitor and a class I HDAC inhibitor (MI-538 and Chidamide) to conventional chemotherapy in t(4;11) pro-B-ALL treatment were ongoing at the time of filing of present application, see detailed description of the experimental setup in Example 10. In analogy to the results shown in Examples 5 to 10 it is expected that the addition of Chidamide will enhance the effect.

In a further embodiment the present invention therefore relates to the composition as disclosed herein above for preventing the relapse of patients having suffered from pro-B- ALL.

As used herein the term "B cell acute lymphoblastic leukemia”, in short “B-ALL”, as used in present invention, refers to acute lymphoblastic leukemia (ALL), a blood cancer that develops from lymphocytes, also called acute lymphocytic leukemia, that develops from B cells.

The term “malignant hematologic cells” as used herein refers to hematologic cancer cells that have their origin in blood-forming tissue, such as the bone marrow, or in the cells of the immune system. Examples of hematologic cancer are leukemia, lymphoma, and multiple myeloma. Hematologic cancer is also called blood cancer.

The term “t(4;11) rearrangement” as used herein refers to a chromosomal anomaly involving a translocation between chromosomes 4 and 11. The chromosomal anomalies are designated by the prefix defining the type of anomaly, in the present case a “t” for translocation. The numbers in brackets indicate the chromosomes involved, i.e. chromosomes 4 and 11 (see e.g. Meyer et al. Leukemia 2006; 20:777-784).

The term “MLL-rearranged hematological disorders” as used herein refers to diseases involving cells of the immunological system, that present genetic alterations at MLL gene. This gene, also known as KMT2A, is located in chromosome 11. A region of the chromosome including this gene is frequently translocated to other chromosomes (such as chromosome 4 or 9) in haematological malignancies, provoking the formation of aberrant fusion proteins that induce a malignant and more aggressive phenotype to cells involved (e.g. Dimartino and Cleary, Br J Haematol 1999). The term “Menin Inhibitor” as used herein relates to inhibitors of Menin, a protein encoded by the MEN1 gene. Menin is a tumor suppressor associated with multiple endocrine neoplasia type 1 (MEN-1 syndrome).

The terms "enhance" or "enhancing" as used herein mean to increase or prolong either in potency or duration a desired effect. By way of example, "enhancing" the effect of therapeutic agents refers to the ability to increase or prolong, either in potency or duration, the effect of therapeutic agents during treatment of a disease, disorder or condition.

The term “Class I HDAC inhibitor” as used herein refers to inhibitors of class I histone deacetylases (HDACs). Histone acetylation levels are maintained by dynamic actions of histone acetyltransferases (HATs), responsible for acetylation, and HDACs, responsible for deacetylation. The HDAC family can be divided into four major classes, each of which consists of different HDAC members: class I (including HDAC 1, 2, 3 and 8), class II (including HDAC 4, 5, 6, 7, 9, 10), class III (including SIRTs 1-7) and class IV (HDAC 11).

It is finally contemplated that any features described herein can optionally be combined with any of the embodiments of any method, medical use, kit and use of a kit of the invention; and any embodiment discussed in this specification can be implemented with respect to any of these. It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention.

All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

The use of the word "a" or "an" may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one". The use of the term “another” may also refer to one or more. The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.

As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. The term “comprises” also encompasses and expressly discloses the terms “consists of” and “consists essentially of”. As used herein, the phrase "consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. As used herein, the phrase "consisting of” excludes any element, step, or ingredient not specified in the claim except for, e.g., impurities ordinarily associated with the element or limitation.

The term "or combinations thereof” as used herein refers to all permutations and combinations of the listed items preceding the term. For example, "A, B, C, or combinations thereof” is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

As used herein, words of approximation such as, without limitation, "about", "around”, “approximately” refers to a condition that when so modified is understood to not necessarily be absolute or perfect but would be considered close enough to those of ordinary skill in the art to warrant designating the condition as being present. The extent to which the description may vary will depend on how great a change can be instituted and still have one of ordinary skilled in the art recognize the modified feature as still having the required characteristics and capabilities of the unmodified feature. In general, but subject to the preceding discussion, a numerical value herein that is modified by a word of approximation such as "about" may vary from the stated value by ±1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10%. Accordingly, the term “about” may mean the indicated value ± 5% of its value, preferably the indicated value ± 2% of its value, most preferably the term “about” means exactly the indicated value (± 0%).

EXAMPLES

MATERIALS AND METHODS

Cell cultures

Human SEM-K2, RS4;11 and REH cell lines were grown and maintained in regular cell culture conditions (RPMI 1640 medium + 10% fetal bovine serum), Where indicated, cells were treated with the respective Menin inhibitor, e.g. MI-463, MI-503, MI-538 or MI-3454, (1 pM, 6 days) and/or Chidamide (1 pM, 48 h). Genetically modified SEM-K2 cells (SEM-TetOn- Tight-HDAC7) were also cultured in RPMI 1640 medium + 10% fetal bovine serum conditions, together with G418 (1.5 mg/mL) and puromycin (0.4 pg/mL) to maintain antibiotic selection. For prednisone treatment experiments, HDAC7 was induced with 1 pg/mL doxycycline 24 h before treating cells with prednisone at 10 pg/mL or 100 pg/mL doses, which was maintained for further 48 h.

RNA extraction and RT-qPCR expression analysis

Total RNA from SEM-K2 cells samples was extracted using Trizol (Life Technologies, Thermo Fisher), following the manufacturer’s instructions. After being quantified, 1-2 pg of RNA were retrotranscribed with random hexamers using a High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, Thermo Fisher). mRNA levels were determined by qRT-PCR using Light Cycler 480 SYBR Green Master Mix (Roche) either in a Light Cycler 480 II (Roche) or in a QuantStudio 7 Flex Real-Time PCR (Applied Biosystems) apparatus. Results were analyzed using LightCycler 480 software (Roche), taking GAPDH and RPL38 as housekeeping genes. All primer pairs were designed with Primer3® software.

Protein extraction and western blot

For protein extraction, SEM-K2, RS4;11 and REH cells were washed with ice-cold PBS and resuspended in RIPA lysis buffer (150 mM NaCI, 50 mM Tris-HCI pH 8.0, 1% NP40, 0.1% SDS, 0.5% sodium deoxycholate) containing Complete Mini EDTA-free protease inhibitors cocktail (Roche), PMSF and DTT. Lysates were sonicated in a UP50H ultrasonic processor (Hielscher), clarified by maximum speed centrifugation and quantified with Bradford reagent (Bio-Rad). Protein lysates were boiled and loaded onto 10% polyacrylamide gels and transferred to 0.2-pm nitrocellulose membranes (Amersham Protran, GE Healthcare Lifescience). After blocking with 5% non-fat milk, membranes were blotted with antibodies against HDAC7 (clone H-273, Santa Cruz Biotechnologies; 1 :500) and p-actin (clone AC-15, Sigma-Aldrich, 1 :2000) overnight at 4°C. After washing with TBS-Tween, membranes were incubated with HRP-conjugated secondary antibodies (anti-mouse HRP and anti-rabbit HRP; 1 :3000) for 1-2 h at r.t. The reaction was developed with the ECL Western Blotting Analysis System (Amersham). Images shown are representative of at least three independent experiments.

MTT cell viability assays

For MTT assays, 5 x 10 ⁴ SEM-K2, RS4;11 and REH cells were seeded in 12-well plates, in triplicate for each condition tested. After the corresponding drug exposition time (MI-463, MI- 503, MI-538, MI-3454, Chidamide or Prednisone) MTT reagent was added at a final concentration of 5 mg/mL. Cells were incubated for 3 h under regular conditions and, afterwards, the resulting formazan blue product was solubilized in DMSO. Absorbance was measured as the difference between absorbance at 570 nm and background absorbance measured at 750 nm. Data are presented as the average of at least three independent experiments, each one of them performed in triplicate.

Example 1 : Upregulation of HDAC7 at protein and mRNA level

A) Treatment of SEM-K2 and RS4;11 cell lines with MI-538 and/or Chidamide

SEM-K2 and RS4;11 cell lines (both harboring t(4; 11) translocation) were treated with MI-538 and/or Chidamide, or DMSO as control, p-actin was used as loading control. Protein levels of HDAC7 in SEM-K2 and RS4;11 cells were determined. The results can be seen in Fig 1A, where the protein levels of HDAC7 in SEM-K2 (left panel) and RS4;11 (right panel) are shown.

B) qRT-PCR for HDAC7 in SEM-K2 cells

SEM-K2 cells were treated with MI-538 and/or Chidamide, or DMSO as control. GAPDH and RPL38 were used as housekeeping genes. Error bars represent SE. Statistical significance is indicated as: * p < 0.05; **, p < 0.01 ; ***, p < 0.001 ; or n.s., non-significant.

A clear upregulation of HDAC7 at protein levels in both cell lines (Figure 1A) and also at mRNA level in SEM-K2 cells (Figure 1 B) could be observed. As can be seen from Figure 1 B, in SEM-K2 cells, treatment with class I HDAC selective inhibitor Chidamide does not provide any increase in mRNA expression of HDAC7, whereas treatment with MI-538 alone leads to an about 1 ,5 fold increase of mRNA expression of HDAC7 in the cells. Surprisingly, the combination of Chidamide and Menin inhibitor MI-538 provides a significant increase in comparison to the use of Chidamide or MI-538 alone in SEM-K2 cells.

As expected, Chidamide did not alter HDAC7 levels in SEM-K2 cells, but slightly increased its expression in RS4;11 (Figure 1A). The combination of both drugs triggered HDAC7 mRNA in SEM-K2 cells (Figure 1 B), converting these combination in a promising strategy for further studies aiming to reduce t(4; 11) pro-B-ALL cells leukemogenic capacity.

C) Quantification of expression of HDAC7 protein

The expression of HDAC7 protein in Figure 1A has been quantified and can be seen in Figure 1 C. Example 2: MTT viability assays

MTT viability assays were conducted in a panel of three pro-B-ALL cell lines, including previously used SEM-K2 and RS4;11 , together with REH cells, that harbor an unaltered form of MLL gene. As expected, REH cells displayed abundant levels of HDAC7, compared to t(4;11) translocated cells (Figure 2A). MI-538 strongly reduced viability of both SEM-K2 and RS4;11 cells, but its combination with Chidamide produced an even more dramatic effect, being significantly stronger than both drugs administered alone (Figure 2B, left and center panels). Interestingly, single therapy with Chidamide presented a low effect in SEM-K2 cells, whereas cell viability reduction was similar to that caused by MI-538 in RS4;11 cell line. This data correlates with the induction of HDAC7 provoked by Chidamide in this specific cell line. The demonstration that the effect of these drugs is, at least partially, a consequence of HDAC7 induction can be extracted from the MTT assays performed in REH cell line. This cell line, with strong expression of HDAC7, did not respond to MI-538 and/or Chidamide treatments, and no effect was observed on cell viability (Figure 2B, right panel).

Example 3: Expression of key HDAC7 targets after induction of HDAC7 with MI-538 and Chidamide

A forced overexpression of HDAC7 in SEM-K2 shifts genomic profile of t(4;11) pro-B-ALL cells towards a more anti-oncogenic profile through the altered expression of targets involved in key hallmarks of cancer, such as cell differentiation, chemotherapy resistance or apoptosis prevention. In order to corroborate whether the effect of MI-538 and Chidamide on endogenous HDAC7 can be paralleled to that of exogenous HDAC7 induction, we analysed some of HDAC7 targets previously reported. qRT-PCR was performed for MMP9 (involved in apoptosis), CD86 (marker of proper B lymphocytes differentiation) and ASNS (chemoresistance marker in infant leukemia) in SEM-K2 cells treated with MI-538 and/or Chidamide, or DMSO as control. Error bars represent SE. Statistical significance is indicated as: * p < 0.05; **, p < 0.01 ; ***, p < 0.001 ; or n.s., non-significant.

According to what the inventors expected, both MI-538 and Chidamide triggered the mRNA levels of the pro-apoptotic gene MMP9 and the marker of B lymphocyte differentiation CD86, showing a synergistic effect when both drugs were administered together (Figure 3, left and center panels). Oppositely, the chemoresistance marker ASNS is downregulated upon treatment of SEM-K2 cells with MI-538 and Chidamide (Figure 3, right panel), according to previous data showing an opposite pattern of expression with HDAC7 (de Barrios et al. 2021). Therefore, the effect of MI-538 and Chidamide not only affects HDAC7 itself, but also its previously described targets, reinforcing the potential use of this combinatorial therapy to improve outcome of t(4; 11) pro-B-ALL. Example 4: Effect of HDAC7 expression on standard therapy

As a proof-of-concept, the effect of HDAC7 expression was evaluated on the regular and standard therapy that is commonly used in clinical settings in infant t(4;11) pro-B-ALL patients. SEM-TetOn-Tight-HDAC7 cell line (also used in de Barrios et al. 2021), that permits HDAC7 induction with doxycycline treatment was used. As shown in Figure 4, HDAC7 induction endows SEM-K2 cells with a higher sensitivity to prednisone treatment, since the reduction of viability is significant at a 10-time lower dose than in normal conditions (i.e. with low HDAC7 levels).

Example 5: MTT viability assays with Menin Inhibitors MI-3454, MI-503 and MI-463

As described in Example 2, the same MTT viability assays were also conducted for other Menin inhibitors MI-3454, MI-503 and MI-463. The results are shown in Fig.5-8. As can be seen in Fig.5, MI-503 shows a strong effect on SEM-K2 cells and the combination with Chidamide shows an even stronger effect, whereas REH cells show only a very weak response to this drug. Fig.6 shows the effect of MI-463, that reduces viability of SEM-K2 cells. However, no additional effect is observed when treated with Chidamide. As expected REH cells do not respond to this inhibitor. Fig.7 shows an effect of Menin inhibitor MI-3454, albeit weaker than the one of MI-503 and MI-463. As can be observed in Fig.8, the use of each of the three Menin inhibitors MI-3454, MI-503 and MI-463 shows an effect on RS4;11 cells, in the case of inhibitor MI-3454 the effect is weaker, but statistically significant.

Example 6: mRNA expression of HDAC7 in SEM-K2 and REH cell lines mRNA expression of HDAC7 in SEM-K2 and REH cell lines has been assessed according to the protocol as described in material the material and methods part. As can be seen in Fig.9 HDAC7 expression increases when SEM-K2 cells are treated with MI-503, MI-463 and MI- 3453 (with or without Chidamide).

Example 7: HDAC7 protein expression in SEM-K2 cells

HDAC7 protein expression in SEM-K2 cells has been assessed according to the protocol as described in material the material and methods part. As can be seen in Fig.10, protein expression of HDAC7 is triggered by MI-503 and MI-463.

Example 8: In vivo experiments in a leukemia murine model

To examine the potential of the therapy with MI-538 alone or in combination with Chidamide for reducing leukemogenesis, in vivo experiments were performed in a leukemia murine model. Immunodeficient NSG mice were used in these assays, in which t(4;11) pro-B-ALL cells kindly obtained from Dr. R.W. Stam (Prinses Maxima Centrum, Utrecht, Netherlands) were used. The procedures involving use of primary cells have been approved by the Research Ethics Committee at Hospital Germans Trias i Pujol (Badalona, Spain), while animal experimentation has been approved by the IRB Committee at CMCiB animal facility (Badalona, Spain).

Before starting the leukemia model, t(4;11) pro-B-ALL cells were expanded and patient- derived xenografts (PDX) generated. For this purpose, 1x10 ⁶ cells were cultured in IM DM medium and, after 24 hours, implanted in NSG immunodeficient mice by intravenous (i.v.) injection. Peripheral blood (PB) was bi-weekly analyzed by flow cytometry, using human CD19, CD45 and HLA-ABC markers, to evaluate engraftment of human cells. When a minimum of 15% of human blasts was reached in PB, mice were euthanized and total cells from bone marrow, spleen and liver were collected and cryopreserved for further use. The percentage of human blasts obtained in the three organs ranged between 65% and 80% of the total cells. These PDX cells have been used to evaluate the use of MI-538 and/or Chidamide as a novel treatment for t(4;11) pro-B-ALL. The experimental procedure is detailed below in Examples 9 and 10.

Example 9: Addition of MI-538 to conventional chemotherapy in t(4; 11) pro-B-ALL treatment PDX cells obtained as described in Example 8 were injected directly in the bone marrow (BM) of NSG mice. Briefly, a total of 18 mice were irradiated at 2 Gy dose and, after 6 h, 1x10 ⁶ PDX cells were implanted by intratibial injection. The engraftment of human pro-B-ALL cells was monitored every 2 weeks by PB extraction and flow cytometry analysis (using the same markers described above). When the percentage of human blasts reached 1% in PB, the percentage of human pro-B-ALL cells in BM was analyzed from BM aspirate samples. As expected, most of the animals presented an engraftment between 10% and 30% in bone marrow. At this point, and previous to treatment initiation, all mice with proper engraftment were split in the different treatment groups, with an equal distribution of the animals to ensure no differences in the average of engraftment between groups. According to BM aspirates data, 6-7 animals were included in each group, leaving 5 untreated animals as control.

The inclusion of MI-538 along with the already defined equivalent to common conventional therapy was tested using the following treatment protocols:

(1) Control group. No treatment

(2) VxL group. Mice treated with two 5-day cycles of the equivalent to conventional therapy, composed by Vincristine (V, 0.15 mg/kg, administered only once weekly), Dexamethasone (x, 5 mg/kg) and L-Asparaginase (L, 1000 lU/kg). Mice were left without treatment for two days between both cycles. (3) VxL + MI-538 group. Mice treated with two VxL cycles as in group (2), but additionally treated with MI-538 at 45 mg/kg during 14 consecutive days.

All treatments were administered intraperitoneally (i.p.)

After two weeks of treatment, PB and BM aspirates were analyzed (Days 15 and 21 posttreatment initiation, respectively) to test whether leukemic cells engraftment had been depleted. Later, mice were followed until day 50 and potential relapse of pro-B-ALL cells was analyzed by weekly PB extraction. Finally, at day 50, mice were euthanized and presence of human leukemic cells in bone marrow, spleen and liver was analyzed.

Example 10: Addition of MI-538 and Chidamide to conventional chemotherapy in t(4; 11) pro- B-ALL treatment

The experiment will be performed in the same way as described for Example 9, but with a total of 32-35 mice, that will be split as follows:

(1) Control group. No treatment

(2) VxL group. Mice treated with two 5-day cycles of the equivalent to conventional therapy, composed by Vincristine (V, 0.15 mg/kg, administered only once weekly), Dexamethasone (x, 5 mg/kg) and L-Asparaginase (L, 1000 lU/kg). Mice are left without treatment for two days between both cycles.

(3) VxL + MI-538 group. Mice treated with two VxL cycles as in group (2), but additionally treated with MI-538 at 45 mg/kg during 14 consecutive days.

(4) VxL + Chidamide group. Mice treated with two VxL cycles as in group (2), but additionally treated with Chidamide at 10 mg/kg administered three times a week.

(5) VxL + MI-538 + Chidamide group. Mice treated with two VxL cycles as in group (2), but additionally treated with MI-538 at 45 mg/kg (for 14 consecutive days) and Chidamide at 10 mg/kg administered three times a week.

All treatments will be administered intraperitoneally (i.p.)

Example 11 : Analysis of HDAC7 expression in diffuse large B cell lymphoma (DLBCL)

63 diffuse large B cell lymphoma (DLBCL) versus 4 healthy Germinal Centre (GC B cells) control samples were used to compare HDAC7 normalized counts obtained by RNA sequencing analysis (figure 18A). Furthermore, a relative HDAC7 mRNA quantification of GC B cells from tonsils (n=4), and DLBCL patients was performed, (see Figure 18B). ABC and GCB refer to two distinct subtypes of DLBCL (n=3, per lymphoma subtype). These analysis clearly showed how HDAC7 is significantly downregulated in DLBCL cells compared to healthy cells derived from germinal centers, i.e., the structure of lymphoid organs from which DLBCL tumors are derived.

Furthermore, data of 292 patients was assessed for overall survival based on HDAC7 expression (Kaplan-Meier plot, see figure 18C). In the figure the grey line corresponds to patients with high HDAC7 expression, and the black line corresponds to patients with low HDAC7 expression. Patients are split in groups by median expression of HDAC7. These data clearly show that DLBCL patients with high HDAC7 expression have a better survival than those with low levels of HDAC7.

Data is represented as mean ± Standard Error (SE) of above-mentioned independent experiments. * p<0.05; ** p<0.01 ; *** p<0.001; unpaired Student’s t test.

Example 12: Role of HDAC7 in DLBCL tumor growth

To assess the role of HDAC7 in DLBCL tumor growth, MD901 cells from DLBCL patients which harbor low levels of HDAC7 were transduced to express HDAC7 in a doxycycline (D) inducible manner (Tet-On-Tet-Tight-HDAC7), and treated, or not, for 72 hours (see Figure 19

A). At the indicated times of treatment with doxycycline, the cell number was determined by cell counting (n=3 per condition).

To assess cell viability, MTT assay were performed with control MD901 cells (E: Tet-On-Tet- Tight-Empty vector) and cells expressing the inducible HDAC7 protein (H7: Tet-On-Tet-Tight- HDAC7) and the absorbance units were measured in indicating cell viability (see Figure 19

B). Where indicated, cells were treated with doxycycline.

It was observed that upon overexpression of exogenous HDAC7 cells stop proliferating and have a much lower viability.

A xenograft assay to assess whether HDAC7 alters tumor growth in DLBCL MD901 cells in vivo was performed on 22 animals (n=11 per condition) as shown in the representative scheme in figure 19C. Photos were taken of the extracted tumors at day 11 post-treatment (figure 19D). A relative volume quantification of tumors and the weight average are shown in figures 19E and F.

In this xenograft in vivo model, it was shown that the induction of HDAC7 drastically reduces tumor growth. Data is represented as mean ± SE of above-mentioned independent experiments. * p<0.05;

** p<0.01 ; *** p<0.001 ; unpaired Student’s t test.

ITEMS OF THE INVENTION

The following embodiments are items of present invention. . A composition, preferably a pharmaceutical composition, for use in a method of inducing expression of HDAC7 in malignant hematologic cells, wherein the composition comprises at least one Menin inhibitor. . The composition for use as described under item 1 , wherein the at least one Menin inhibitor is a compound according to formula (A) wherein

Ri is a C1-4 alkyl group including branched alkyl optionally substituted with up to three halogen atoms;

R2 is H or NRaRb, Ra and Rb are independently H or C1-4 alkyl;

Ra is H or OH;

R4 independently represents H, C1-4 alkyl group including cycloalkyl, or aryl group including heteroaryl;

R4 is optionally substituted with Re, where Re represents H, NRcRd and Rc and Rd are independently H, C1-4 alkyl group including branched alkyl optionally substituted with up to three halogen atoms, CHO or CONRcRd n is 0 - 4

Rs is H, or a C1-4 alkyl group including branched alkyl optionally substituted with up to three halogen atoms. The composition of item 2, wherein

Ri is -CH2CF3

R ₂ is H or MeNH-

R3 is H or OH

R4 independently represents H, a 4-pyrazolyl group (I) or a substituted bicyclic alkyl group n is 0 - 4

Rs is H or methyl. The composition of items 1 to 3, wherein

R1 is -CH2CF3

R ₂ is H

R3 is H or OH

R4 is H or a 4-pyrazolyl group - n is 0 or 1

Rs is H or methyl. The composition of items 1 to 4, wherein

R1 is -CH2CF3

R2 and Rs are H

R ₃ is OH

R4 is a 4-pyrazolyl group - The composition for use according to any one of items 1 to 5, wherein the at least one Menin inhibitor is selected from MI-538, MI-503, MI-463, MI-3454, preferably wherein the Menin inhibitor is MI-538.

7. The composition for use according to any one of items 1 to 6, wherein the expression of HDAC7 is mRNA expression and wherein said mRNA expression of HDAC7 is increased by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold in the malignant hematologic cells compared to the mRNA expression of HDAC7 in reference cells.

8. The composition for use according to any one of items 1 to 7, wherein the expression of HDAC7 is protein expression and wherein said protein expression of HDAC7 is increased by at least about 1.5-fold, 2.0-fold, 2.5-fold, 3.0-fold, 3.5-fold, 4.0-fold, 4.5- fold, 5.0-fold, 5.5-fold, 6.0-fold, 6.5-fold, 7.0-fold, 7.5-fold, 8.0-fold, 8.5-fold, 9.0-fold, 9.5-fold, 10.0-fold, 10.5-fold, 11.0-fold, 11.5-fold, 12.0-fold in the malignant hematologic cells compared to the protein expression of HDAC7 in reference cells.

9. The composition for use according to any one of items 1 to 8, wherein the malignant hematologic cells are selected from T-cell acute lymphoblastic leukemia (T-ALL) or B cell derived hematologic cells, preferably B cell acute lymphoblastic leukemia (B-ALL) cells, diffuse large B cell lymphoma (DLBCL) cells and follicular lymphoma (FL), more preferably wherein the B-ALL cells are B cell acute lymphoblastic leukemia (B-ALL) cells.

10. The composition for use according to any one of items 1 to 9, wherein the B-ALL cells are pro-B-all cells characterized by a t(4; 11) rearrangement.

11. The composition for use according to any one of items 1 to 10, wherein the composition further comprises at least one Class I HDAC inhibitor.

12. The composition for use according to item 11, wherein the at least one Class I HDAC inhibitor is selected from the group consisting of Chidamide, Entinostat, Mocetinostat, Tacedinaline and Romidepsin, or a combination thereof.

13. The composition for use according to item 12, wherein the at least one Class I HDAC inhibitor is Chidamide. 14. The composition for use according to any of items 11 to 13, wherein the at least one Menin inhibitor is MI-538 and the at least one Class I HDAC inhibitor is Chidamide.

15. A kit of parts comprising at least two recipients, wherein one recipient comprises at least one Menin inhibitor and the other recipient comprises at least one Class I HDAC inhibitor according to any one of the preceding claims.

16. The kit of parts according to item 15, wherein the at least one Menin inhibitor is selected from the group consisting of MI-538, MI-503, MI-463 or MI-3454, preferably wherein the at least one Menin inhibitor is MI-538.

17. The kit of parts according to any of items 15 or 16, wherein the at least one Class I HDAC inhibitor is selected from the group consisting of Chidamide, Entinostat Mocetinostat, Tacedinaline and Romidepsin, or a combination thereof, preferably wherein the at least one Class I HDAC inhibitor is Chidamide.

18. The kit of parts according to any of items 15 to 17, wherein the at least one Menin inhibitor is MI-538 and the at least one Class I HDAC inhibitor is Chidamide.

19. The composition according to any one of items 1 to 14 and the kit of parts of claims 15 to 18 for use in a method of treatment of T-cell acute lymphoblastic leukemia (T- ALL), B cell acute lymphoblastic leukemia (B-ALL), diffuse large B cell lymphoma (DLBCL) or follicular lymphoma (FL), preferably of B cell acute lymphoblastic leukemia (B-ALL).

20. A method for treating T-cell acute lymphoblastic leukemia (T-ALL), B cell acute lymphoblastic leukemia (B-ALL), diffuse large B cell lymphoma (DLBCL) or follicular lymphoma (FL), preferably B cell acute lymphoblastic leukemia (B-ALL) in a subject in need thereof, comprising administering to said subject the composition as defined in items 1 to 14.