Stemness-associated cell states are linked to chemotherapy resistance in AML. We uncovered a direct mechanistic link between expression of the stem cell transcription factor GATA2 and drug resistance. The GATA-binding protein 2 (GATA2) plays a central role in blood stem cell generation and maintenance. We find substantial intra- and inter-patient variability in GATA2 expression across AML patient samples. GATA2 expression varies by molecular subtype and has been linked to outcome. In a murine model, KMT2A-MLL3 driven AML originating from a stem cell or immature progenitor cell population have higher Gata2 expression and are more resistant to the standard AML chemotherapy agent doxorubicin. Deletion of Gata2 resulted in more robust induction of p53 following exposure to doxorubicin. ChIP-Seq, RNA-Seq and functional studies revealed that GATA2 regulates the expression of RASSF4, a modulator of the p53 inhibitor MDM2. GATA2 and RASSF4 are anti-correlated in human cell lines and AML patient cell bulk and single cell expression datasets. Knockdown of Rassf4 in Gata2 low cells resulted in doxorubicin or nutlin-3 resistance. Conversely, overexpression of Rassf4 results in sensitization of cells expressing high levels of Gata2. Finally, doxorubicin and nutlin-3 are synergistic in Gata2-high murine AML, as well as AML patient samples. We discovered a previously unappreciated role for GATA2 in dampening p53-mediated apoptosis via transcriptional regulation of RASSF4, a modulator of MDM2. This role for GATA2 directly links the expression of a stemness associated transcription factor to chemotherapy resistance.

Inhibitors of the menin-KMT2A interaction are promising agents for the treatment of KMT2A-rearranged leukemias. We evaluated menin inhibition in patient-derived xenografts of KMT2A-rearranged leukemias with high-risk features. Three acute myeloid leukemias with high-risk fusion partners (mixed-lineage leukemia-10 [MLLT10] and mixed-lineage leukemia-4 [MLLT4]) and two infant acute lymphocytic leukemia (ALL) samples were sensitive to menin inhibition. We also evaluated serial samples from two patients with multiply relapsed ALL. We found that highly pretreated KMT2A::AFF1 ALL samples were much less sensitive compared with cells obtained earlier in the same patients' disease course. Because none of the patients had been treated with a menin inhibitor, resistance in these highly pretreated samples was acquired in the absence of menin-inhibitor exposure. Transcriptomic analysis documented sustained on-target efficacy toward the canonical targets of the menin inhibitor in resistant cells. Targeted genomic analysis documented the emergence of multiple comutations, including RAS pathway and TP53 mutations, although neither was sufficient to induce menin-inhibitor resistance in vitro. Downregulation of KMT3D may account for resistance in one patient; inactivation of KMT2C/D has been reported to result in menin-inhibitor resistance, and KMT2C-edited cells from this patient were selected for in menin-inhibitor-containing growth conditions. Future studies will need to clarify more broadly which genomic/epigenomic alterations drive upfront resistance. Regardless of mechanism, our data support using menin inhibitors upfront or in early lines of therapy before substantial genomic or epigenomic evolution has occurred.

Stem cell transcriptional signatures are linked to poor outcomes in AML. Conventional chemotherapy (+/-HSCT) remains the only curative approach for AML, and survival largely reflects chemotherapy sensitivity. The mechanism by which a stem-cell-like transcriptional signature promotes chemotherapy resistance and relapse has not been determined. We identified a direct link between stemness and p53-mediated apoptosis involving a GATA2-RASSF4-MDM2-p53 axis. To interrogate transcriptional states that might mediate resistance in AML patients, we conducted single-cell RNA sequencing and single-cell ATAC sequencing on 10 pediatric AML samples. We found that expression of the stem/progenitor cell transcription factor GATA2 identified an immature cell population that partially overlaps with populations characterized by ERG and MECOM expression and chromatin accessibility. Using a Gata2 conditional retroviral KMT2A-MLLT3 mouse model, we established a model to study functional consequences of distinct GATA2 expression levels. This analysis revealed that AML originating from a murine stem cell or immature progenitor cell population showed higher Gata2 expression and were more resistant to standard AML chemotherapy agents. While GATA2 was not strictly required for leukemogenesis, Gata2high cells were more resistant to doxorubicin than Gata2low cells. In vitro treatment of bulk leukemias with doxorubicin resulted in selection for Gata2high cells. GATA2 expression was also increased in patient AML samples at relapse compared to initial diagnosis. Genetic inactivation of Gata2 profoundly enhanced chemosensitivity in Gata2high, but not Gata2low leukemias. Deletion of Gata2 in Gata2high cells increased activation of p53-mediated apoptosis in response to nutlin-3. Importantly, loss of Gata2 decreased MDM2 protein stability indicating that GATA2 may blunt the p53 response by enhancing MDM2 stabilization. ChIP-Seq and RNA-Seq in murine KMT2A-MLLT3 leukemias showed that neither p53 nor Mdm2 are transcriptionally regulated by GATA2. We next sought to identify a potential regulator of MDM2 stability. Integrated RNA-Seq and ChIP-Seq data nominated Rassf4, which was significantly upregulated upon Gata2 ablation, and GATA2 occupied its promoter. Similar to leukemia cells, decreasing Gata2 expression in normal hematopoietic stem cells increased Rassf4 expression, a pattern that is partially rescued by re-expressing Gata2. The RASSF family has been shown to promote apoptosis by modulating MDM2 protein stability. We functionally validated whether the relationship between high Gata2 expression and drug resistance is mediated by Rassf4 repression. Gata2-/- MA9 cells transduced with Rassf4 sgRNAs gained a competitive growth advantage under the pressure of nutlin-3a treatment versus cells transduced with non-targeting sgRNA indicating the emergence and expansion of drug-resistant cells. Conversely, overexpressing Rassf4 in Gata2high MA9 cells sensitized them to nutlin-3a and chemotherapy. Furthermore, GATA2 and RASSF4 expression inversely correlated in our AML patient sample scRNA-Seq dataset, as well as publicly available AML patient datasets, supporting the notion that a GATA2 mechanism to suppress RASSF4 applies to multiple AML subtypes. We evaluated whether MDM2 inhibitors, such as Idasanutlin, in combination with doxorubicin, could overcome the drug resistance seen in Gata2high leukemias. Combination therapy prolonged survival in vivo. In conclusion, we identified a novel role for GATA2 in blunting p53-mediated apoptosis via transcriptional repression of Rassf4, a regulator of MDM2 protein stability. Our study supports a model where the “volume control” of p53-mediated apoptosis by a stem cell transcription factor is an integral part of stemness, which is imparted on leukemic cells arising from a stem-cell-like cell-of-origin. Our findings provide a mechanistic explanation for the well-established, but thus far unexplained observation that the expression of HSC signatures are associated with poor outcomes in AML.

Inhibitors of the Menin-KMT2A interaction are emerging as promising agents for the treatment of KMT2A-rearranged ( KMT2A-r) acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). Menin is required for the recruitment of the KMT2A-fusion to a subset of its target genes. Early phase clinical trials have shown highly encouraging results in patients with relapsed and refractory leukemias with KMT2A rearrangements and NPM1 mutations. In a recently reported early phase clinical trial evaluating the Menin inhibitor revumenib, about 50% of patients failed to show a clinical response despite robust downregulation of known Menin:KMT2A target genes, suggesting on target activity of the drug. This was the most common pattern of upfront resistance. We sought to understand upfront resistance (in contrast to the reported induced resistance via MEN1 mutations) in patient derived xenograft models. To this end, we evaluated the efficacy of Menin-inhibition in KMT2A-r leukemias with high-risk features: high-risk KMT2A fusion partners in AML ( MLLT10, MLLT4), infant ALL, and serial samples from patients with multiply relapsed ALL. We found that AML samples with high-risk fusion partners banked at initial diagnosis were sensitive to Menin-inhibition in xenografts. In contrast, three highly pretreated samples from patients with KMT2A-AFF1 ALL showed only a slight, or no decrease in latentcy in the Menin-inhibitor arm. Interestingly, Menin inhibition showed excellent in vivo efficacy against leukemia samples obtained from the same three patients earlier in their disease course. Therefore, prior chemotherapy (+/- immunotherapy) selects for Menin-inhibitor resistant cells without prior exposure to Menin inhibitor. Transcriptomic analysis (RNA-Seq) documents sustained on-target efficacy of the Menin inhibitor on a transcriptional level in resistant cells. None of the samples contained a MEN1 mutation at any time point. Targeted panel sequencing at initial diagnosis and relapse documented clonal evolution and the emergence of multiple co-mutations (particularly RAS pathway mutations and TP53 mutations), however, all co-mutations tested failed to induce resistance as a single alteration. It is possible that the cooperation of multiple emerging mutations (rather than a single mutation) is sufficient to drive AML in a KMT2A-fusion independent manner, thus mediating Menin resistance. A transcriptomic response (i.e. downregulation of KMT2A-fusion target genes) without a clinical response was the most common pattern of upfront resistance in the recently reported phase I/II clinical trial of the Menin inhibitor revumenib. We conclude that it will be critical to use Menin-inhibitors upfront or in early lines of therapy before substantial clonal evolution has occurred.

Inhibitors of the Menin-KMT2A interaction are emerging as promising agents for the treatment of KMT2A -rearranged ( KMT2A -r) acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). Menin is required for the recruitment of the KMT2A -fusion to a subset of its target genes. We evaluated the efficacy of Menin-inhibition in KMT2A-r leukemias with high-risk features: high-risk KMT2A fusion partners in AML ( MLLT10, MLLT4 ), infant ALL, and serial samples from patients with multiply relapsed ALL. We find that AML with high-risk fusion partners is sensitive to Menin-inhibition in xenografts. In contrast, highly pretreated samples from patients with KMT2A-AFF1 ALL are largely resistant. Menin inhibitor resistance in these patients is acquired, as Menin inhibition shows in vivo efficacy against leukemia samples obtained earlier in the same patients’ disease course. Transcriptomic analysis documents sustained on-target efficacy of the Menin inhibitor on a transcriptional level in resistant cells. However, genomic analysis documented the emergence of multiple co-mutations that may be sufficient to drive AML, and may mediate independence from the KMT2A-fusion induced transcription program. Our data suggest that it will be critical to use Menin-inhibitors upfront or in early lines of therapy before substantial clonal evolution has occurred.

Long term survival from AML reflects in large parts the ability to completely eradicate all AML cells. In most patients an initial remission can be achieved, but a small number of drug resistant cells often survive and mediate relapse. Both genetic (i.e. subclonal mutational heterogeneity) and epigenetic mechanisms likely play a role in the emergence of resistance. In order to interrogate transcriptional states within bulk AML patient samples that might mediate epigenetic resistance, we conduced single cell RNA-Seq on 10 pediatric AML samples at initial diagnosis. Results were confirmed by analyzing publicly available data from 12 adult AML samples. We found surprisingly large heterogeneity in GATA2 expression both intra- and inter-patient. GATA2 is a transcription factor widely expressed throughout the hematopoietic system, with predominant expression in hematopoietic stem cells (HSCs) and myeloid progenitors. The role of GATA2 in pathogenesis of AML is complex, with overexpression, as well as gain, altered and loss of function mutations of GATA2 reported in AML. Several independent studies reported that high GATA2 expression is associated with worse outcomes. Intra-patient, GATA2 high expressing cells (GATA2high) skewed more immature, although both GATA2high and GATA2low cells were found at nearly all stages of maturation within the AML. When comparing both GATA2high and GATA2low cells within the same leukemia, pathways related to ribosomal biogenesis (RiBi) and protein translation were the most enriched in GATA2high subclones. For functional studies, we turned to a Gata2 conditional retroviral KMT2A-MLLT3 mouse model. Similar to the patient samples, this model also showed both variable expression of Gata2 among different leukemic mice as well as within leukemia cells from the same mouse. Both high Gata2 (Gata2high) and low Gata2 (Gata2low) expressing clones were able to engraft and cause leukemia in recipient mice, although Gata2low clones did so with slightly lower penetrance and slightly longer latency. Similarly, deletion of Gata2 in bulk leukemia resulted in lower penetrance and longer latency, however, we found multiple animals with fully deleted, transplantable Gata2-/-AML showing that Gata2 is not absolutely required for leukemogenesis. Deletion of Gata2 from Gata2high murine AML cells resulted in significant downregulation of RiBi and protein synthesis pathways. Cut&Run identified binding of Gata2 at promoters of genes encoding ribosome proteins. This suggests a direct regulation of RiBi and protein homeostasis by Gata2. Multiple recent reports have implicated ribosomal proteins in the regulation of p53 mediated apoptosis. Indeed, we found that Gata2high murine AML cells showed a blunted p53 stabilization in response to the Mdm2 inhibitor nutlin-3, and Gata2high were more resistant to doxorubicin than Gata2low AML cells. Furthermore, in vitro treatment of bulk leukemias with Doxorubicin resulted in selection for Gata2high cells. Deletion of Gata2 sensitized leukemia cells to nutlin-3 and chemotherapy. We next treated murine AML cells with the Pol1 inhibitor CX-5461, which inhibits RiBi. While Gata2high AML cells had been more resistant to doxorubicin, they were more sensitive to CX-5461 than Gata2low AML cells. In addition, we observed profound synergy between CX-5461 and doxorubicin. In conclusion, we identified Gata2high subclones in patients with AML that likely represent a reservoir for resistance and relapse. Our functional data document high RiBi and a blunted p53 response in Gata2high AML cells that is dependent on Gata2. This cell state can be successfully targeted with a combination of standard chemotherapy and a Pol I inhibitor, nominating this combination as a promising therapeutic avenue for patients with AML.

Self-renewal of spermatogonial stem cells is vital to lifelong production of male gametes and thus fertility. However, the underlying mechanisms remain enigmatic. Here, we show that DOT1L, the sole H3K79 methyltransferase, is required for spermatogonial stem cell self-renewal. Mice lacking DOT1L fail to maintain spermatogonial stemcells, characterized by a sequential loss of germ cells from spermatogonia to spermatids and ultimately a Sertoli cell only syndrome. Inhibition of DOT1L reduces the stem cell activity after transplantation. DOT1L promotes expression of the fate-determining HoxC transcription factors in spermatogonial stem cells. Furthermore, H3K79me2 accumulates at HoxC9 and HoxC10 genes. Our findings identify an essential function for DOT1L in adult stem cells and provide an epigenetic paradigm for regulation of spermatogonial stem cells.

Acute graft-versus-host disease (aGvHD) is a severe and often life-threatening complication of allogeneic hematopoietic cell transplantation (allo-HCT). AGvHD is mediated by alloreactive donor T-cells targeting predominantly the gastrointestinal tract, liver, and skin. Recent work in mice and patients undergoing allo-HCT showed that alloreactive T-cells can be identified by the expression of α4β7 integrin on T-cells even before manifestation of an aGvHD. Here, we investigated whether the detection of a combination of the expression of T-cell surface markers on peripheral blood (PB) CD8 + T-cells would improve the ability to predict aGvHD. To this end, we employed two independent preclinical models of minor histocompatibility antigen mismatched allo-HCT following myeloablative conditioning. Expression profiles of integrins, selectins, chemokine receptors, and activation markers of PB donor T-cells were measured with multiparameter flow cytometry at multiple time points before the onset of clinical aGvHD symptoms. In both allo-HCT models, we demonstrated a significant upregulation of α4β7 integrin, CD162E, CD162P, and conversely, a downregulation of CD62L on donor T-cells, which could be correlated with the development of aGvHD. Other surface markers, such as CD25, CD69, and CC-chemokine receptors were not found to be predictive markers. Based on these preclinical data from mouse models, we propose a surface marker panel on peripheral blood T-cells after allo-HCT combining α4β7 integrin with CD62L, CD162E, and CD162P (cutaneous lymphocyte antigens, CLA, in humans) to identify patients at risk for developing aGvHD early after allo-HCT.

Internal tandem duplication mutations in the Fms-like tyrosine kinase 3 (FLT3-ITD) are frequently recurring in AML and confer a poor prognosis. FLT3 inhibitors (FLT3i) such as gilteritinib are efficacious in relapsed AML. Clinical responses to FLT3i include myeloid differentiation of the FLT3-ITD clone in about 50% of patients. How FLT3i induce this response in a subset of patients is unknown.

Meningioma-1 (MN1) overexpression in AML is associated with poor prognosis, and forced expression of MN1 induces leukemia in mice. We sought to determine how MN1 causes AML. We found that overexpression of MN1 can be induced by translocations that result in hijacking of a downstream enhancer. Structure predictions revealed that the entire MN1 coding frame is disordered. We identified the myeloid progenitor-specific BAF complex as the key interaction partner of MN1. MN1 over-stabilizes BAF on enhancer chromatin, a function directly linked to the presence of a long polyQ-stretch within MN1. BAF over-stabilization at binding sites of transcription factors regulating a hematopoietic stem/progenitor program prevents the developmentally appropriate decommissioning of these enhancers and results in impaired myeloid differentiation and leukemia. Beyond AML, our data detail how the overexpression of a polyQ protein, in the absence of any coding sequence mutation, can be sufficient to cause malignant transformation.

Translocations of Meningioma-1 (MN1) occur in a subset of acute myeloid leukemias (AML) and result in high expression of MN1, either as a full-length protein, or as a fusion protein that includes most of the N-terminus of MN1. High levels of MN1 correlate with poor prognosis. When overexpressed in murine hematopoietic progenitors, MN1 causes an aggressive AML characterized by an aberrant myeloid precursor-like gene expression program that shares features of KMT2A-rearranged (KMT2A-r) leukemia, including high levels of Hoxa and Meis1 gene expression. Compounds that target a critical KMT2A-Menin interaction have proven effective in KMT2A-r leukemia. Here, we demonstrate that Menin (Men1) is also critical for the self-renewal of MN1-driven AML through the maintenance of a distinct gene expression program. Genetic inactivation of Men1 led to a decrease in the number of functional leukemia-initiating cells. Pharmacologic inhibition of the KMT2A-Menin interaction decreased colony-forming activity, induced differentiation programs in MN1-driven murine leukemia and decreased leukemic burden in a human AML xenograft carrying an MN1-ETV6 translocation. Collectively, these results nominate Menin inhibition as a promising therapeutic strategy in MN1-driven leukemia.

ABSTRACT Inhibition of the histone methyl-transferase DOT1L (KMT4) has shown encouraging activity in preclinical models of KMT2A ( MLL) -rearranged leukemia. The DOT1L inhibitor pinometostat (EPZ5676) was well tolerated in early phase clinical trials and showed modest clinical activity, including occasional complete responses (CRs) as single agent. These studies support the development of combinatorial therapies for KMT2A -rearranged leukemias. Here, we investigated two novel combinations: dual inhibition of the histone methyltransferases DOT1L and EZH2, and the combination of a DOT1L inhibitor with the protein synthesis inhibitor homoharringtonine (HHR). EZH2 is the catalytic histone methyltransferase in the polycomb repressive complex 2 (PRC2), and inhibition of EZH2 has reported preclinical activity in KMT2A -rearranged leukemia. We found that the H3K79 and H3K27 methyl marks are not dependent on each other, and that DOT1L and EZH2 inhibition affect largely distinct gene expression programs. In particular, the KMT2A/DOT1L target HOXA9, which is commonly de-repressed as a consequence of PRC2 loss or inhibition in other contexts, was not re-activated upon dual DOT1L/EZH2 knockout or inhibition. Despite encouraging data in murine KMT2A-MLLT3 transformed cells suggesting synergy between DOT1L and EZH2 inhibition, we found both synergistic and antagonistic effects on a panel of human KMT2A rearranged cell lines. Combinatorial inhibition of DOT1L and EZH2 is thus not a promising strategy. We identified opposing effects on ribosomal gene transcription and protein translation by DOT1L and EZH2 as a mechanism that is partially responsible for observed antagonistic effects. The effects of DOT1L inhibition on ribosomal gene expression prompted us to evaluate the combination of EPZ5676 with a protein translation inhibitor. EPZ5676 was synergistic with the protein translation inhibitor homoharringtonine (HHR), supporting further preclinical/clinical development of this combination.

Understanding mechanisms of cooperation between oncogenes is critical for the development of novel therapies and rational combinations. Acute myeloid leukemia (AML) cells with KMT2A-fusions and KMT2A partial tandem duplications (KMT2APTD) are known to depend on the histone methyltransferase DOT1L, which methylates histone 3 lysine 79 (H3K79). About 30% of KMT2APTD AMLs carry mutations in IDH1/2 (mIDH1/2). Previous studies showed that 2-hydroxyglutarate produced by mIDH1/2 increases H3K79 methylation, and mIDH1/2 patient samples are sensitive to DOT1L inhibition. Together, these findings suggested that stabilization or increases in H3K79 methylation associated with IDH mutations support the proliferation of leukemias dependent on this mark. However, we found that mIDH1/2 and KMT2A alterations failed to cooperate in an experimental model. Instead, mIDH1/2 and 2-hydroxyglutarate exert toxic effects, specifically on KMT2A-rearranged AML cells (fusions/partial tandem duplications). Mechanistically, we uncover an epigenetic barrier to efficient cooperation; mIDH1/2 expression is associated with high global histone 3 lysine 79 dimethylation (H3K79me2) levels, whereas global H3K79me2 is obligate low in KMT2A-rearranged AML. Increasing H3K79me2 levels, specifically in KMT2A-rearrangement leukemias, resulted in transcriptional downregulation of KMT2A target genes and impaired leukemia cell growth. Our study details a complex genetic and epigenetic interaction of 2 classes of oncogenes, IDH1/2 mutations and KMT2A rearrangements, that is unexpected based on the high percentage of IDH mutations in KMT2APTD AML. KMT2A rearrangements are associated with a trend toward lower response rates to mIDH1/2 inhibitors. The substantial adaptation that has to occur for 2 initially counteracting mutations to be tolerated within the same leukemic cell may provide at least a partial explanation for this observation.

Meningioma-1 (MN1) was first described in a sporadic t(4;22) translocation in meningioma. Rare MN1 fusions to the ETS factors ETV6 (t(12;22)) and FLI1 (cryptic) also occur in AML. t(12;22) translocations have been described in two different patterns:Type 1: MN1 exon 1, is fused to a C-terminal fragment of ETV6. The first exon codes for almost all of the protein and has been shown previously to be sufficient to induce AML in a mouse model. Type 2: The entire MN1 coding frame, including the stop codon, is fused to a portion of ETV6. This results in a fusion on a DNA level, but on a protein level only MN1 is expressed. In both cases the fusion results in very high MN1 expression, either alone (type 2) or as a fusion protein (type 1). We performed ChIP-seq for H3K4me2/H3K27ac/Med1 on cell lines and a primary patient sample, and identified a large putative enhancer within and downstream of the ETV6 locus. Type 2 translocations suggest that hijacking of this enhancer is the critical oncogenic event. Besides the rare fusions, a subgroup of AML patients have very high MN1 expression without evident fusions. We speculate that some of these patients may have cryptic enhancer fusions. Importantly, several independent studies show that MN1 overexpression confers a poor prognosis. The survival rate 2 years after diagnosis is at only 20-30% reflecting the aggressiveness of this leukemia. In mice MN1 overexpression induces one of the most aggressive leukemias known as a single hit. These leukemias are Hoxa9 high and transcriptionally resemble KMT2A-rearranged leukemias. Despite its clear contribution to aggressive AML, it is not understood how MN1 functions on a molecular level. MN1 has no identified classic structural domains and lacks sequence homology with any other protein. Therefore, no predictions about structure or possible binding partners exist, and only few binding partners have been shown experimentally. This severely limits therapy options for patients and potential future drug development. Therefore, we aimed to define the MN1 interaction partner(s) and mechanism of leukemogenesis. To identify the MN1 interactome in AML we used two complementary methods, co-immunoprecipitation (CoIP) and proximity-dependent labeling (BioID), followed by Mass Spectrometry. As top hit in both screens we identified the mSWI/SNF complex, including the ATPase Smarca4, as an interactor of MN1. mSWI/SNF is a multisubunit complex with cell context- and function- dependent variable members. This complex is responsible for chromatin remodeling and plays an important role in gene expression and lineage determination. Its role in various forms of cancer is well established, where subunits are deleted, mutated, or misrecruited. We find co-sedimentation of MN1 with identified mSWI/SNF members in glycerol gradients using murine and human cells with MN1 overexpression. ChIP-seq data indicates a high overlap in DNA occupancy between MN1 and Smarca4. Using a conditional Smarca4 KO mouse model we show that Smarca4 is indispensable for MN1 driven leukemia. Together, these experiments substantiate a critical interaction between the oncogenic driver MN1 and the epigenetic modifier complex mSWI/SNF. MN1 contains a long polyQ stretch encoded by 28 CAG repeats. Such glutamine rich regions have been recognized as domains facilitating transcriptional activation, stabilizing protein-protein interactions, and being important components in higher order complex formation. PolyQ domains belong to the family of prion-like domains, which have roles in SWI/SNF recruitment. We show that the deletion of MN1's polyQ stretch abolishes differentiation block and allows the cells to differentiate. This is reflected in poor replating efficiency in Methylcellulose assays compared to full length MN1 driven leukemia cells. In vivo, MN1s' polyQ stretch is important for AML initiation. On a molecular level, we find that polyQ deletion reduces the affinity of the mSWI/SNF complex to chromatin in comparison to full length MN1, and fails to maintain the expression of key MN1 target genes such as the later Hoxa cluster, Meis1 and Flt3. In conclusion, our data support a model wherein MN1's oncogenic function is mediated by mSWI/SNF dysregulation, via the MN1 polyQ stretch. Disclosures Bernt: Glaxo-Smith-Kline: Other: Family member working for GSK; Agios: Consultancy; Epizyme: Other: applied for joint patent.

Inhibition of the histone methyl-transferase DOT1L (KMT4) has shown encouraging activity in preclinical models of KMT2A (MLL)-rearranged leukemia. The DOT1L inhibitor pinometostat (EPZ5676) was well tolerated in early phase clinical trials and showed modest clinical activity, including occasional complete responses (CRs) as single agent. These studies support the development of combinatorial therapies for KMT2A-rearranged leukemias. Here, we investigated two novel combinations: dual inhibition of the histone methyltransferases DOT1L and EZH2, and the combination of a DOT1L inhibitor with the protein synthesis inhibitor homoharringtonine (HHR). EZH2 is the catalytic histone methyltransferase in the polycomb repressive complex 2 (PRC2), and inhibition of EZH2 has reported preclinical activity in KMT2A-rearranged leukemia. We found that the H3K79 and H3K27 methyl marks are not dependent on each other, and that DOT1L and EZH2 inhibition affect largely distinct gene expression programs. In particular, the KMT2A/DOT1L target HOXA9, which is commonly de-repressed as a consequence of PRC2 loss or inhibition in other contexts, was not re-activated upon dual DOT1L/EZH2 knockout or inhibition. Despite encouraging data in murine KMT2A-MLLT3 transformed cells suggesting synergy between DOT1L and EZH2 inhibition, we found both synergistic and antagonistic effects on a panel of human KMT2A rearranged cell lines. Combinatorial inhibition of DOT1L and EZH2 is thus not a promising strategy. We identified opposing effects on ribosomal gene transcription and protein translation by DOT1L and EZH2 as a mechanism that is partially responsible for observed antagonistic effects. The effects of DOT1L inhibition on ribosomal gene expression prompted us to evaluate the combination of EPZ5676 with a protein translation inhibitor. EPZ5676 was synergistic with the protein translation inhibitor homoharringtonine (HHR), supporting further preclinical/clinical development of this combination.

Acute graft versus host disease (aGVHD) remains a major complication in patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT), the only curative treatment for many malignant hematologic diseases. After initial priming in secondary lymphoid organs, alloreactive donor T cells efficiently migrate to the intestinal tract, liver and skin. We observed that alloreactive effector T cells infiltrating and attacking the lamina propria of the small and large intestines closely interact with intestinal myeloid cells of host origin. Here we asked whether these intimate interactions regulate alloreactive effector T cell responses and how they impact intestinal aGvHD.

Meningioma-1 (MN1) has been found overexpressed in acute myeloid leukemia (AML). High MN1 expression levels are associated with poor prognosis and limited therapeutic options. Overexpression of MN1 in murine bone marrow progenitor cells causes leukemia as a single hit. Interestingly, MN1 induces a gene expression program that is reminiscent of KMT2a-rearranged leukemia, with high expression of the KMT2a (MLL-1) target genes HOXA7-13 and MEIS1. However, MN1 oncogenic functions remain unclear and no targetable therapies are available for MN1 high leukemia. We have previously shown, using a conditional knock-out mouse, that deletion of the histone methyltransferase Kmt2a increased latency and decreased penetrance of MN1 driven leukemia. We sought to determine what specific function of KMT2A is relevant to MN1 leukemogenesis in order to identify new therapeutic targets.

Rationale: KMT2A-rearrangements (KMT2A-r) in acute myeloid leukemia (AML), encompassing both KMT2a-fusions (KMT2A-F) and KMT2A-partial tandem duplications (KMT2A-PTD), represent a subgroup of AML with a particularly poor prognosis. Both KMT2A-F and KMT2A-PTD share a dependency on the H3K79 methyltransferase DOT1L for proper histone 3 lysine 79 dimethylation (H3K79me2) on target genes such as HOXA9 and MEIS1. Pharmacologic inhibition of DOT1L results in downregulation of KMT2A fusion/PTD target genes. Albeit rare, complete responses observed in patients with relapsed/refractory KMT2A-r leukemia treated with a DOT1L inhibitor underscore the clinical relevance of this pathway. The second most commonly co-occurring mutation with KTM2A-PTD are mutations in isocitrate dehydrogenase 1 and 2 (mIDH1/2). The canonical oncogenic function of mIDH1/2 involves aberrant production of the oncometabolite 2HG and inhibition of TET2. However, 2HG also induces histone hypermethylation, including H3K79 hypermethylation as reported by several groups. Based on this, we sought to study the effect of increasing H3K79 methylation in KMT2A-r AML either via direct overexpression of DOT1L or introduction of mIDH1/2.