Menin Inhibition Research Articles

NPM1-fusion proteins promote myeloid leukemogenesis through XPO1-dependent HOX activation.

Nucleophosmin (NPM1) is a nucleolar protein and one of the most frequently mutated genes in acute myeloid leukemia (AML). In addition to the commonly detected frameshift mutations in exon12 (NPM1c), previous studies have identified NPM1 gene rearrangements leading to the expression of NPM1-fusion proteins in pediatric AML. However, whether the NPM1-fusions are indeed oncogenic and how the NPM1-fusions cause AML have been largely unknown. In this study, we investigated the subcellular localization and leukemogenic potential of two rare NPM1-fusion proteins, NPM1::MLF1 and NPM1::CCDC28A. NPM1::MLF1 is present in both the nucleus and cytoplasm and occasionally induces AML in the mouse transplantation assay. NPM1::CCDC28A is more localized to the cytoplasm, immortalizes mouse bone marrow cells in vitro and efficiently induces AML in vivo. Mechanistically, both NPM1-fusions bind to the HOX gene cluster and, like NPM1c, cause aberrant upregulation of HOX genes in cooperation with XPO1. The XPO1 inhibitor selinexor suppressed HOX activation and colony formation driven by the NPM1-fusions. NPM1::CCDC28A cells were also sensitive to menin inhibition. Thus, our study provides experimental evidence that both NPM1::MLF1 and NPM1::CCDC28A are oncogenes with functions similar to NPM1c. Inhibition of XPO1 and menin may be a promising strategy for the NPM1-rearranged AML.

Epigenetic regulation of noncanonical menin targets modulates menin inhibitor response in acute myeloid leukemia

AbstractMenin inhibitors that disrupt the menin-MLL interaction hold promise for treating specific acute myeloid leukemia (AML) subtypes, including those with KMT2A rearrangements (KMT2A-r), yet resistance remains a challenge. Here, through systematic chromatin-focused CRISPR screens, along with genetic, epigenetic, and pharmacologic studies in a variety of human and mouse KMT2A-r AML models, we uncovered a potential resistance mechanism independent of canonical menin-MLL targets. We show that a group of noncanonical menin targets, which are bivalently cooccupied by active menin and repressive H2AK119ub marks, are typically downregulated after menin inhibition. Loss of polycomb repressive complex 1.1 (PRC1.1) subunits, such as polycomb group ring finger 1 (PCGF1) or BCL6 corepressor (BCOR), leads to menin inhibitor resistance by epigenetic reactivation of these noncanonical targets, including MYC. Genetic and pharmacological inhibition of MYC can resensitize PRC1.1-deficient leukemia cells to menin inhibition. Moreover, we demonstrate that leukemia cells with the loss of PRC1.1 subunits exhibit reduced monocytic gene signatures and are susceptible to BCL2 inhibition, and that combinational treatment with venetoclax overcomes the resistance to menin inhibition in PRC1.1-deficient leukemia cells. These findings highlight the important roles of PRC1.1 and its regulated noncanonical menin targets in modulating the menin inhibitor response and provide potential strategies to treat leukemia with compromised PRC1.1 function.

Revumenib for patients with acute leukemia: a new tool for differentiation therapy.

Treatment of acute leukemia is gradually moving away from a "one-size-fits-all" approach, as scientific and clinical advances expand the arsenal of available targeted therapies. One of the recent additions are the menin inhibitors; oral, selective, small molecules that disrupt the interaction between the chromatin adapter menin, and an epigenetic regulator, the Lysine Methyltransferase 2A (KMT2A) complex. Two susceptible leukemia subtypes have been identified: 1) Acute Myeloid Leukemia (AML) with a mutation in Nucleophosmin 1 (NPM1), and 2) any acute leukemia, myeloid or lymphoid, with a translocation resulting in the rearrangement of KMT2A. These leukemias share a distinct genetic expression, maintained by the KMT2A-menin interaction. Together they account for approximately 40% of patients with AML, and 10% of patients with Acute Lymphoblastic Leukemia (ALL). This review follows the journey of revumenib, as a representative of menin inhibitors, from bench to bedside. It will focus on the pathophysiology of leukemias sensitive to menin inhibition, delineation of how this understanding led to targeted drug development, and data from clinical trials. The important discovery of resistance mechanisms will also be explored, as well as future directions in using menin inhibitors for treating leukemia.

Combination of menin and kinase inhibitors as an effective treatment for leukemia with NUP98 translocations.

Chromosomal translocations of the nucleoporin 98 (NUP98) gene are found in acute myeloid leukemia (AML) patients leading to very poor outcomes. The oncogenic activity of NUP98 fusion proteins is dependent on the interaction between Mixed Lineage Leukemia 1 and menin. NUP98-rearranged (NUP98-r) leukemia cells also rely on specific kinases, including CDK6 and/or FLT3, suggesting that simultaneous targeting of these kinases and menin could overcome limited sensitivity to single agents. Here, we found that combinations of menin inhibitor, MI-3454, with kinase inhibitors targeting either CDK6 (Palbociclib) or FLT3 (Gilteritinib) strongly enhance the anti-leukemic effect of menin inhibition in NUP98-r leukemia models. We found strong synergistic effects of both combinations on cell growth, colony formation and differentiation in patient samples with NUP98 translocations. These combinations also markedly augmented anti-leukemic efficacy of menin inhibitor in Patient Derived Xenograft models of NUP98-r leukemia. Despite inhibiting two unrelated kinases, when Palbociclib or Gilteritinib were combined with the menin inhibitor, they affected similar pathways relevant to leukemogenesis, including cell cycle regulation, cell proliferation and differentiation. This study provides strong rationale for clinical translation of the combination of menin and kinase inhibitors as novel treatments for NUP98-r leukemia, supporting the unexplored combinations of epigenetic drugs with kinase inhibitors.

Abstract PR012: KAT6A/B and Menin-MLL complexes coordinately regulate estrogen receptor-driven gene expression programs in breast cancer

Abstract The estrogen receptor (ER) is expressed in ∼70% of breast cancers where it functions as a critical transcription factor that regulates cell growth and tumor progression. ER inhibition is a mainstay treatment for ER-positive (ER+) breast cancer, but despite the clinical benefit of targeting ER signaling, breast cancer remains the second leading cause of cancer-related death in women, with ∼50% of deaths resulting from ER+ tumors. While recurrent and metastatic ER+ tumors are typically resistant to endocrine therapies they continue to rely on ER signaling suggesting that disruption of other chromatin associated complexes that mediate ER-driven gene expression may present an alternative therapeutic strategy. One chromatin modifier important in regulating ER function is the histone acetyltransferase KAT6A. KAT6A is amplified and/or overexpressed in ∼15% of ER+ tumors and a novel KAT6A/B inhibitor has entered clinical trials for advanced breast cancer. However, as with most single agent therapies, compensatory mechanisms may attenuate the effects of KAT6A/B inhibition on ER-driven gene expression. We hypothesized that the dual targeting of distinct chromatin complexes would enhance the effects of KAT6A/B inhibition, by altering chromatin state and transcriptional output. To that end we performed an epigenetic focused CRISPR-Cas9-based functional genetic screen to identify chromatin-associated proteins that enhance KAT6A/B inhibition. CRISPR guides targeting MEN1 (encoding Menin) were among our top hits as sensitizers to treatment with the KAT6A/B inhibitor PF-9363. We confirmed that co-treatment of PF-9363, and the Menin inhibitor, SNDX-5613, had strong synergistic anti-proliferative effects in a panel of ER+ breast cancer cell lines. Mechanistically, our data revealed that KAT6A/B and Menin cooperatively regulate ER-driven gene expression and chromatin accessibility, both through direct effects on ESR1 expression as well as through effects at ER target genes. Specifically, we found that KAT6A and Menin-KMT2A complexes co-localized at promoters of ER target genes and treatment with KAT6A/B or Menin inhibitors displaced KAT6A and Menin/KMT2A from ER target genes, respectively. Yet only the combination effectively displaced both complexes from chromatin, which produced more dramatic changes in ER-directed gene expression than either small molecule alone. Combined displacement of KAT6A and Menin/KMT2A at ER-regulated genes induced loss of an active chromatin landscape characterized by reduction of activating histone modifications and loss of RNA Polymerase II binding to ER target loci. Importantly, the combination of KAT6A/B and Menin inhibition was effective in a panel of ER+ patient-derived organoid models as well as in various models of endocrine resistance, including models with ESR1 mutations, NF1 loss, and FOXA1 mutations. Both KAT6A/B and Menin inhibitors are in Phase I/II clinical trials and have shown manageable toxicity profiles to date. This combination represents a novel potential therapeutic combination for ER+ breast cancer. Citation Format: Sarah Naomi Olsen, Bryn Anderson, Charles Hatton, Rinath Jeselsohn, Myles Brown, Eneda Toska, Scott Armstrong. KAT6A/B and Menin-MLL complexes coordinately regulate estrogen receptor-driven gene expression programs in breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr PR012.

Distinct Responses to Menin Inhibition and Synergy with DOT1L Inhibition in KMT2A-Rearranged Acute Lymphoblastic and Myeloid Leukemia.

Pediatric acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) exhibit favorable survival rates. However, for AML and ALL patients carrying KMT2A gene translocations clinical outcome remains unsatisfactory. Key players in KMT2A-fusion-driven leukemogenesis include menin and DOT1L. Recently, menin inhibitors like revumenib have garnered attention for their potential therapeutic efficacy in treating KMT2A-rearranged acute leukemias. However, resistance to menin inhibition poses challenges, and identifying which patients would benefit from revumenib treatment is crucial. Here, we investigated the in vitro response to revumenib in KMT2A-rearranged ALL and AML. While ALL samples show rapid, dose-dependent induction of leukemic cell death, AML responses are much slower and promote myeloid differentiation. Furthermore, we reveal that acquired resistance to revumenib in KMT2A-rearranged ALL cells can occur either through the acquisition of MEN1 mutations or independently of mutations in MEN1. Finally, we demonstrate significant synergy between revumenib and the DOT1L inhibitor pinometostat in KMT2A-rearranged ALL, suggesting that such drug combinations represent a potent therapeutic strategy for these patients. Collectively, our findings underscore the complexity of resistance mechanisms and advocate for precise patient stratification to optimize the use of menin inhibitors in KMT2A-rearranged acute leukemia.

The Menin story in acute myeloid leukaemia-The road to success.

The treatment of acute myeloid leukaemia (AML) has changed fundamentally in the last decade with many new targeted therapies entering clinics. Some of the most interesting agents under development are Menin inhibitors which interfere with the interaction of Menin with wild-type (wt) KMT2A or a KMT2A-fusion protein and thereby downregulate the leukaemic gene expression (MEIS1, PBX3, HOX) in NPM1 mutant or KMT2A-rearranged leukaemia. Other HOX and MEIS1 expressing leukaemias may also be sensitive to Menin inhibition. Following the encouraging results as monotherapy in refractory and relapsed AML, the combination of Menin inhibitors with chemotherapeutic agents and other targeted drugs is being investigated clinically.

Synergistic Effects of the RARalpha Agonist Tamibarotene and the Menin Inhibitor Revumenib in Acute Myeloid Leukemia Cells with KMT2A Rearrangement or NPM1 Mutation.

Inhibition of menin in acute myeloid leukemia (AML) harboring histone-lysine-N-methyltransferase 2A rearrangement (KMT2Ar) or the mutated Nucleophosmin gene (NPM1c) is considered a novel and effective treatment approach in these patients. However, rapid acquisition of resistance mutations can impair treatment success. In patients with elevated retinoic acid receptor alpha (RARA) expression levels, promising effects are demonstrated by the next-generation RARalpha agonist tamibarotene, which restores differentiation or induces apoptosis. In this study, the combination of revumenib and tamibarotene was investigated in various KMT2Ar or NPM1c AML cell lines and patient-derived blasts, focusing on the potential synergistic induction of differentiation or apoptosis. Both effects were analyzed by flow cytometry and validated by Western blot analysis. Synergy calculations were performed using viability assays. Regulation of the relevant key mediators for the MLL complex were quantified by RT-qPCR. In MV4:11 cells characterized by the highest relative mRNA levels of RARA, highly synergistic induction of apoptosis is demonstrated upon combination treatment. Induction of apoptosis by combined treatment of MV4:11 cells is accompanied by pronounced induction of the pro-apoptotic protein BAX and a synergistic reduction in CDK6 mRNA levels. In MOLM13 and OCI-AML3 cells, an increase in differentiation markers like PU.1 or a decreased ratio of phosphorylated to total CEBPA is demonstrated. In parts, corresponding effects were observed in patient-derived AML cells carrying either KMT2Ar or NPM1c. The impact of revumenib on KMT2Ar or NPM1c AML cells was significantly enhanced when combined with tamibarotene, demonstrating synergistic differentiation or apoptosis initiation. These findings propose promising strategies for relapsed/refractory AML patients with defined molecular characteristics.

Abstract 577: Deciphering the mechanism of the menin-MLL complex dependency in HCC

Abstract Liver cancer is the third leading cause of cancer-related death worldwide, with hepatocellular carcinoma (HCC) being the most common primary liver cancer (~90%). The 5-year survival rate of patients with advanced HCC is ~18% due to the late prognosis and the moderate clinical benefit of available systemic therapy options. Mutations in chromatin regulators were detected in ~50% of liver cancer. Since many chromatin regulators have been shown a dependency in various cancer types and can modulate therapy response, we focused on identifying new therapeutic targets of HCC among epigenetic modifiers and evaluating them as potential targets for future drug development. We constructed a CRISPR library of 6000 gRNAs targeting 737 genes involved in chromatin-mediated gene regulation. Using the epigenome-focused CRISPR/Cas9 screening in two-dimensional and three-dimensional settings in multiple liver cancer cell lines (HepG2, HLF, PLC/PRF5), we identified a list of epigenetic regulators as potential new targets in HCC. Two of them, MEN1 and ASH2L, are core subunits of the menin-MLL complex mediating H3K4 trimethylation (me3). We validated dependencies of HCC on MEN1 and ASH2L by performing a negative selection CRISPR/Cas9 competitive growth assay in HLF and PLC/PRF5 cell lines. Moreover, treatment of HCC cell lines (HLF, PLC/PRF5, HepG2) with recently developed menin inhibitor SNDX-5613 (revumenib) revealed a dose-dependent reduction in cell proliferation. To determine transcriptional changes associated with disruption of the menin-MLL complex, we performed RNA sequencing of HLF and PLC/PRF5 cells following either inhibition with 5µM SNDX-5613 for 4 days or knockout of the MEN1 or ASH2L gene. We observed the upregulation of KRAS and the oncogenic signature of TGFβ signaling pathways in all menin-MLL complex disrupting conditions. We did not detect global changes in H3K4me3 levels upon the menin inhibition or MEN1 or ASH2L knockouts assessed by immunoblotting. To explore the mechanism of the antiproliferative effect of the menin inhibition on HCC further, we evaluated changes in the binding of menin to chromatin and H3K4me3 levels in HCC cells by performing CUT&RUN following treatment of HLF cells with 5µM SNDX-5613 for 4 days. Menin inhibition led to a drastic removal of menin from the chromatin, while H3K4me3 levels were only mildly decreased. We identified 8765 genes near promoter regions with menin binding and H3K4me3 mark, of which 1952 genes were near regions with simultaneous loss of menin and decreased H3K4me3 levels upon menin inhibition. Integration of our genomics data revealed a list of 30 genes, which are downregulated upon menin inhibition in both HLF and PLC/PRF5 cells (FC≥1.5) and the direct targets of the menin-MLL complex in HLF cells, suggesting their potential role in HCC cell survival. Altogether, we anticipate that menin and ASH2L serve as promising targets and represent an appealing therapeutic strategy for HCC treatment. Citation Format: Margarita M. Dzama, Peyton C. Kuhlers, Jesse R. Raab. Deciphering the mechanism of the menin-MLL complex dependency in HCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 577.

Mezigdomide is effective alone and in combination with menin inhibition in preclinical models of KMT2A-r and NPM1c AML

AbstractSmall molecules that target the menin-KMT2A protein-protein interaction (menin inhibitors) have recently entered clinical trials in lysine methyltransferase 2A (KMT2A or MLL1)–rearranged (KMT2A-r) and nucleophosmin-mutant (NPM1c) acute myeloid leukemia (AML) and are demonstrating encouraging results. However, rationally chosen combination therapy is needed to improve responses and prevent resistance. We have previously identified IKZF1/IKAROS as a target in KMT2A-r AML and shown in preclinical models that IKAROS protein degradation with lenalidomide or iberdomide has modest single-agent activity yet can synergize with menin inhibitors. Recently, the novel IKAROS degrader mezigdomide was developed with greatly enhanced IKAROS protein degradation. In this study, we show that mezigdomide has increased preclinical activity in vitro as a single-agent in KMT2A-r and NPM1c AML cell lines, including sensitivity in cell lines resistant to lenalidomide and iberdomide. Further, we demonstrate that mezigdomide has the greatest capacity to synergize with and induce apoptosis in combination with menin inhibitors, including in MEN1 mutant models. We show that the superior activity of mezigdomide compared with lenalidomide or iberdomide is due to its increased depth, rate, and duration of IKAROS protein degradation. Single-agent mezigdomide was efficacious in 5 patient-derived xenograft models of KMT2A-r and 1 NPM1c AML. The combination of mezigdomide with the menin inhibitor VTP-50469 increased survival and prevented and overcame MEN1 mutations that mediate resistance in patients receiving menin inhibitor monotherapy. These results support prioritization of mezigdomide for early phase clinical trials in KMT2A-r and NPM1c AML, either as a single agent or in combination with menin inhibitors.

Transcriptomic without Clinical Response to Menin Inhibition As a Mechanism of Upfront Resistance in Samples from Mutliply Relapsed Patients with KMT2A-Rearranged Leukemia

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.

Single-Cell Proteomic Analysis Reveals Menin Inhibition-Induced Proteomic Alterations in AML Patients Treated with Revumenib

Menin inhibition is a promising therapeutic approach for acute myeloid leukemia (AML) with certain genetic alterations including KMT2A rearrangements or NPM1 mutations. The first-in-human Phase 1 clinical trial of revumenib (SNDX-5613) in patients with R/R AML showed promising results and induced complete remission in patients who refractory to multiple lines to therapy. These findings suggest that revumenib could provide a new therapeutic option for patients with AML with KMT2A and NPM1 genetic alterations. AML is characterized by the existence of distinct differentiation hierarchies, a phenomenon substantially contributing to disease heterogeneity. AML hierarchies exhibit distinct expressions of phenotypic profiles, apoptotic networks, signaling pathways, and metabolic profiles, leading to varying sensitivity towards targeted therapies, resulting in differential sensitivity to targeted therapies. Revumenib induces AML differentiation and thus shift the balance between AML hierarchies, resulting in drastic alterations in the overall AML proteomic profiles and enrichment of distinct leukemia phenotypes with differential sensitivity to targeted therapies. Detailed interrogation of revumenib-induced proteomic alterations could permit precise targeting of persistent cells and development of data-driven combinatorial approaches to achieve higher response rates. Moreover, the distinct combinations of genetic alterations can impact the efficacy of revumenib-induced differentiation and therapeutic outcomes. Thus, to better understand these complexities we performed multimodal single-cell (sc) analysis of AML landscape in R/R AML treated with revumenib (n=4) in Phase 1. We utilized two leukemia-focused 51-parameter CyTOF panels to analyze pre and post-treatment samples from R/R AML patients treated with revumenib. Sc proteogenomic profiling was performed using MissionBio platform using a panel covering 37 genes with recurrent AML-relevant mutations. UMAP and clustering analyses of CyTOF data identified AML blasts, unveiled quantitative changes in cell composition, and delineated proteomic shifts induced under revumenib therapy pressure. We observed enrichment of CD11b+/-CD68+, more differentiated monocytic cells situated at close proximity to AML blasts and depletion of immature AML blasts. Through unsupervised analysis, we discovered discrete AML differentiation hierarchies, delineated by a differentiation continuum that initiates with immature AML blasts (CD34+/-c-Kit+/-), progresses through intermediate AML blasts (CD68+/CD11b-/CD14-), and culminates with more differentiated monocytic cells (CD68++/CD11b+/CD14+/-). Notably, intermediate AML blasts and monocytic cells were enriched in post-treatment samples. Simultaneous ScDNA+protein analysis showed enrichment of differentiated AML blasts with monocytic/dendritic cell phenotype and harboring AML mutations. Differentiated AML blasts expressed higher levels of MCL1 and BCL-xL and upon differentiation we observed gradual downregulation of BCL2. Importantly, intermediate and more differentiated AML blasts under revumenib therapy expressed high levels of p53 protein, a feature that can partly attributed to NPM1 mutations. Sc proteomic analysis delineated proteomic shifts elicited by revumenib and identified potential therapeutic partners, by targeting MCL1, BCL-xL and MDM2, that could be used in combination with revumenib to improve therapeutic efficacy. We next performed differential expression analysis by comparing the proteomic profiles of pre- and post-therapy samples. We observed suppression of less differentiated AML phenotypic profiles (c-Kit, CD123, CD34) along with MEIS1 and PBX3. Importantly, we also observed significant alterations in immune landscape, suggesting that revumenib could rewire Myeloid-to-T and NK cell interaction/communication by inducing proteomic shifts and driving AML blast differentiation. Further analysis of an expanded cohort of R/R AML patients is currently underway. In conclusion, multimodal sc analysis revealed profound proteomic alterations induced by revumenib and identified potential therapeutic targets. These findings present new avenues for optimizing combinatorial treatment regimens, enhancing revumenib's efficacy, and illuminating survival mechanisms in persistent, differentiated AML blasts.

Targeting CDK9 in KMT2A-Rearranged Infant Leukemia: Evidence for Activity and Drug Synergy with Enitociclib

Introduction: Translocations of the lysine methyltransferase 2A (KMT2A) gene on 11q23 are seen in most infant leukemias. The outcomes in cases with KMT2A rearrangements (KMT2Ar) are significantly worse due to higher disease relapse rates and the emergence of chemo-resistance. Studies have shown that P-TEFb (CDK9/Cyclin T1) is an important component of the KMT2Ar complex and drives the transcriptional elongation step, subsequently contributing to the development of leukemia (Mueller, Dorothee et al.,2009). Based on this rationale, we investigated the potential of the selective P-TEFb/CDK9 inhibitor enitociclib in infant leukemia. Enitociclib potently inhibits MYC and MCL-1 in various tumor models (Sher, Steven et al.,2023). In addition, menin has been shown to be an important protein in the activation of target genes by KMT2Ar fusions, and menin inhibitors are considered promising therapeutic agents against infant leukemias, with at least four menin inhibitors currently in clinical development. We show preclinical data to support the activity of enitociclib with respect to cellular cytotoxicity, apoptosis induction and drug synergy in infant leukemia cells. Methods: A panel of pediatric leukemia cell lines representing high-risk and treatment resistant KMT2Ar cells were used. Leukemia cells with wild-type KMT2A as well as normal CD34+ cells and PBMCs from healthy donors were used as controls. Leukemic cells were treated with enitociclib (0.5µM - 0.03µM) and cell viability was measured using alamar blue assay. Apoptotic cell death following enitociclib treatment was measured by Annexin V/Propidium Iodide flow cytometry. Target modulation analyses were carried out by western blotting. Bone marrow (BM) stroma and leukemia co-culture experiments were performed to identify the effect of enitociclib on BM-derived growth support provided to leukemic cells. Drug combination assays were carried out with the menin inhibitor MI-463 and combination indices were calculated using the Chao-Talalay method. Rat xenograft models bearing infant leukemia cells were used to study the efficacy and tolerability of enitociclib therapy. Results: Data from in vitro cytotoxicity assays showed high sensitivity of KMT2Ar infant leukemia cells to enitociclib with IC 50 values of 30nM for KMT2Ar cells and 406nM for KMT2A-WT cells. Annexin V/PI staining showed a dose-dependent increase in cells in the late apoptotic stage for KMT2Ar cells after treatment. Target validation assays in KMT2Ar cells showed inhibition of transcriptional elongation by decreased serine phosphorylation by approximately 75% of RNA POL II CTD starting as early as 6 hours after treatment with 0.1µM enitociclib. Additionally, in KMT2Ar cells, an 80%-85% reduction in the amount of MCL-1 and MYC protein levels was observed after treatment with 0.1µM enitociclib for 6 hours. Furthermore, our data show that enitociclib may help to overcome the BM derived stromal mediated survival advantage in KMT2Ar leukemic cells. Enitociclib also potentiated the anti-leukemic effect of chemotherapies for childhood leukemia with the most effective combination observed with doxorubicin (0.01µM IC 50 for doxorubicin alone, 0.002µM IC 50 when combined with enitociclib) and prednisolone (25µM IC 50 for prednisolone only, 0.01µM IC 50 in combination). Drug combination assays showed effective synergy between enitociclib and the KMT2A-menin inhibitor MI-463 in KMT2Ar infant leukemia cells with a combination index of less than 1. As a detectable biomarker of activity, HOXA9 protein levels were found to be robustly diminished with 60% decrease after 24-hour treatment with the drug combination. In vivo, enitociclib given once weekly for 3 weeks led to complete remission in 10 out of 12 rats bearing KMT2Ar xenografts. Discussion: Our data demonstrate the ability of enitociclib to induce effective cellular cytotoxicity in high-risk KMT2Ar leukemia cells. We further show that enitociclib potentiated the anti-leukemic effect of chemotherapies that constitute the backbone of childhood leukemia protocols. Importantly, we show synergy with menin inhibition that is currently in consideration to be included in new generation clinical trials, suggesting the effective use of this novel combination in future treatment protocols. We provide the first in vitro and animal data supporting enitociclib in future treatment approaches for infants with KMT2Ar leukemia.

Purine Metabolism Modulates Leukemia Stem Cell Maintenance in MLL-Rearranged Acute Leukemia

Acute myeloid leukemia (AML) represents the most prevalent hematopoietic malignancy driven by leukemia stem cells (LSCs) and remains challenging to treat in adult patients. MLL rearrangement results in a genetically unique subtype of AML characterized by chromosomal translocations involving the MLL gene and over 80 fusion partners, including AF9 as one of the most common partners. The oncogenic MLL fusion proteins interact with multiple transcription and chromatin regulators, such as Menin, to induce leukemogenic stem cell gene programs and transform hematopoietic stem and progenitor cells. Although significant progress has been made in understanding the metabolic dysregulation in acute leukemia, such as alterations in glycolysis, energetic metabolism, amino acid metabolism, and fatty acid metabolism, the metabolic factors that modulate MLL fusion oncogenic transcriptional activity in AML remain incompletely elucidated. To identify novel metabolic vulnerabilities in MLL-rearranged AML, we performed a whole genome CRISPR dropout screen and profiled the metabolites of murine LSCs of MLL-AF9 murine model. This analysis revealed the purine biosynthesis pathway is essential for MLL-rearranged leukemia and that MLL-rearranged LSCs are enriched in metabolites of the purine biosynthesis pathway. Isotypoe incorporation experiments using [U- 13C]glucose demonstrated a high purine biosynthetic rate in the LSCs compared to bulk leukemia cells. Accordingly, human acute myeloid leukemia stem cells express high levels of the purine biosynthetic genes. Furthermore, we found that MYC, a well-known downstream target of MLL-AF9, mediates the enhanced purine synthesis in LSCs. These results demonstrate enhanced purine biosynthetic pathways in LSCs. We utilized genetic and pharmacological (mycophenolate mofetil, MMF, a purine biosynthesis inhibitor of IMPDH) approaches to target the enhanced purine metabolism and found inhibition of the purine synthesis pathway promotes myeloid differentiation of both murine and human LSCs. Metabolomics analysis reveals a remarkable reduction of purine metabolites. Supplementing the LSCs with guanosine, an intermediate metabolite of the purine biosynthesis pathway, substantially rescued the differentiation. Importantly, treatment of MLL-AF9-driven murine leukemia mice with MMF impaired LSCs function, prolonged leukemia survival, and reduced leukemia burden in the peripheral blood and bone marrow, while causing minimal effects on normal hematopoiesis. These results demonstrate the reliance on enhanced purine metabolism in LSCs maintenance. Previous work has shown that the purine biosynthesis pathway is essential for rRNA transcription in the nucleolus, which could be inhibited by CX-5461. To verify the essentiality of the rRNA transcription in MLL-rearranged LSCs, we treated MLL-AF9-induced murine LSCs cells with CX-5461 and found CX-5461 promotes myeloid differentiation of LSCs. Moreover, we found that the myeloid differentiation induced by MMF or CX-5461 is associated with reduced chromatin occupancy of the MLL-AF9 complex, especially Menin, and downregulated expression of MLL-AF9 target genes, such as Meis1, Hoxa9 and Myc, suggesting a regulatory role of nucleolar rRNA transcription on MLL-AF9 oncogenic gene expression program. We are currently focusing on the synergistic effects of targeting purine metabolism and Menin inhibition on AML suppression. Altogether, our findings reveal that purine metabolism maintains nucleolar rRNA transcription homeostasis to modulate MLL fusion complex-induced leukemogenic transcriptional activity in LSCs. The enhanced purine metabolism emerges as a crucial dependency for LSCs, providing potential targets for novel therapeutic strategies in treating MLL-rearranged leukemia.

The LSD1 Inhibitor Ory-1001 (ladademstat) in Combination with Menin Inhibitor SNDX-5613 (revumenib) Has Synergistic in Vitro Activity in KMT2A-Rearranged AML Models

Introduction: Menin is a scaffold protein that binds to gene promoters, enhancers and transcription factors, thereby influencing transcription. Leukemias with rearrangement of KMT2A known as mixed-lineage leukemia (MLL), as well as other abnormalities such as NPM1c, respond to Menin inhibition with promising early clinical results. MLL leukemias affect children and adults and are associated with high rates of resistance to conventional chemotherapy. In addition to these alterations, other leukemia subsets with similar transcriptional dependency could be targeted through Menin inhibition. LSD1 is an essential regulator of murine MLL-AF9 leukemia stem cells (LSCs) and maintains the LSC potential of MLL-AML cells through blocking of differentiation and apoptosis ( Harris et al, 2012). LSD1 acts at genomic loci that are bound by MLL-AF9 to maintain expression of the associated leukemogenic program, thus preventing differentiation and apoptosis. We investigated the pre-clinical efficacy of combined Menin and LSD1 inhibition in AML. PC4 and SF2 interacting protein 1 (PSIP1)/p75, also known as LEDGF, interacts with the N-terminus of MLL and Menin. This complex contributes to the association of MLL and MLL-fusion multiprotein complexes with the chromatin. Several studies have shown that both PSIP1 and Menin are required for efficient MLL-fusion-mediated leukemogenesis and for the expression of MLL-regulated genes. A proposed model of LSD1 interaction with KMT2A-Menin-PSIP1 suprcomplex is depicted in Fig. 1A. Methods: To assess in vitro LSD1 inhibitor (ORY-1001) and Menin inhibitor (SNDX) potency, cell lines were plated and treated with the respective doses of ORY-1001, SNDX, and the combination of these drugs in 96-well plates. We performed synergy assays using increasing concentrations of SNDX and 3µM of ORY-1001. Cell viability and IC50 were measured after 4 days of treatment. The SynergyFinder 2.0 web application was used to calculate synergy scores and generate 2D synergy maps. Western blotting of LSD1, co-immunoprecipitation, and ChIP-qPCR studies were used to assess the depletion of the LSD1 from chromatin. The interaction network of LSD1 interactome proteins was assessed by mass spectrometry. Results: We showed that LSD1 degrades with increasing concentrations of ORY-1001 after 48h of treatment (Fig.1 B). IC 50 curves for ORY-1001, SNDX and synergy effect is shown with increasing concentrations of SNDX and 3µM ORY-1001 in MV4-11 cells (expressing MLL-AF4) (Fig.1 C). ORY-1001 alone shows an IC 50 of ~0.5 µM, and SNDX alone shows an IC 50 of ~10 nM. The combination of LSD1 and SNDX inhibitors synergistically reduced cell viability in MLL-rearranged MOLM-13 and MV4-11 cells (Fig.1 C). We also assayed the LSD1 endogenous protein interactome in MOLM-13 cells (expressing MLL-AF9) by mass spectrometry. Our LSD1 interactome analysis revealed an association of LSD1 with previously identified interactors such as RCOR1, HDAC1, HDAC2, CARM1 and HMG20B. We discovered an unexpected association between LSD1 and PSIP1. Interestingly, PSIP1 interact with MLL1-MEN1 complex and its crucial for assembling MLL-fusion proteins to their gene targets. Conclusion: LSD1 Inhibitor ladademstat in Combination with Menin Inhibitor revumenib has synergistic effect in KMT2A-Rearranged AML models. Surprisingly, we also found that PSIP1, which is essential for inducing MLL-rearranged leukemia, interacts with LSD1. This suggests that PSIP1 may modulate gene expression through its ability to interact with both MLL1 and LSD1. This study opens a new avenue to investigate the role of KMT2A-MEN1 in relation to other epigenetic modifiers, such as LSD1, and guide new potential combination therapies that may further potentiate differentiation and elimination of leukemia LSCs.

Novel Inhibitors of MLLT1/3, a Critical Component of the Super-Elongation Complex (SEC), Target a Range of Molecularly Defined Acute Leukemias, with a Differential Profile to Menin Inhibition

Introduction: The SEC is a developmental transcriptional regulator which is often hijacked by cancer. The SEC regulates RNA Polymerase II dependent transcription of major oncogenes e.g., Myc, Bcl2, CDK6, along with developmental genes e.g. HOXA9 and MEIS1. SEC-dependent transcription is recognised as a driver in multiple acute myeloid leukemia (AML) sub-populations, such as those defined by Mixed Lineage Leukemia 1 (MLL1)-rearrangements (MLLr), which are prevalent in >10% of acute leukemias, and NPM1 mutations, which account for ~30% of leukemias. MLLT1 is known to be a key nucleating component of the SEC, and CRISPR/Cas-9 screens have previously highlighted a critical dependency for MLLT1 in MLLr driven leukemias. Clinical proof-of-concept for targeting the SEC has recently been demonstrated with inhibitors of menin, a protein that also impacts SEC activity in certain contexts. However, whilst the clinical data with menin inhibitors have been encouraging, there are a number of AML cell lines and patients that do not respond, and a growing number of examples of acquired clinical resistance. Hence there is a need for additional approaches which may benefit a broader spectrum of patients. Targeting MLLT1/3 provides one such promising approach. We have discovered novel MLLT1/3 inhibitors which have broad therapeutic potential across a range of molecularly defined acute leukemias, with a differential profile to menin inhibitors. Methods: In this study, a FRET assay was used to measure the ability of MLLT inhibitors to prevent the interaction of the MLLT1 and MLLT3 YEATS domains with acetylated histones. Selectivity was assessed against other YEATS domain proteins. MLLT target gene expression was measured by qPCR in leukemia cell lines. The anti-proliferative potential of MLLT1/3 inhibitors was assessed across a broad range of leukemia and normal cell lines and compared with menin inhibitors and other standard of care compounds using a cell viability readout (total ATP quantification). Combination studies were also performed in leukemia cell lines with MLLT1/3 and menin inhibitors. The clonogenic potential of MLLT1/3 inhibitors was determined using colony formation assays. Apoptosis was measured using caspase 3/7 readout. In vivo activity was assessed in a MV4;11 subcutaneous model. Results: We have identified novel MLLT1/3 inhibitors which block the interaction of the MLLT1 and 3 YEATS domains with acylated histones with IC 50 values <100 nM. They were shown to have excellent selectivity for MLLT1/3, with no inhibition observed against two other YEATS domain proteins, YEATS2 and 4. The MLLT1/3 inhibitors were shown to inhibit the expression of MLLT target genes known to be regulated via the SEC e.g., MYC and HOXA9. These compounds profoundly inhibited the growth of MLLr fusion and NPM1 mutant leukemias, with minimal activity seen against normal cell lines. Notably, potent inhibition of cell growth in non-MLLr fusion leukemia cell lines was observed, which were not sensitive to menin inhibition. MLLT1/3 inhibitors were shown to induce apoptosis and block the clonogenic potential of leukemia cell lines. Synergy between MLLT1/3 and menin inhibitors was also observed in leukemic cell lines. MLLT1/3 inhibitors have been tested in an MV4;11 model with substantial tumour growth inhibition observed. Conclusion: Targeting MLLT1/3 is an attractive approach and has the potential for single agent activity across a broad range of molecular defined acute leukemias, with a differential profile to menin inhibitors.

Decoding the Epigenetic Drivers of Menin-MLL Inhibitor Resistance in KMT2A-Rearranged Acute Myeloid Leukemia

Menin inhibitors that disrupt Menin-MLL interaction are candidate therapeutic agents for several acute myeloid leukemia (AML) subtypes such as KMT2A rearrangement and NPM1 mutation. Several Menin inhibitors are currently being assessed in clinical trials and have demonstrated promising initial results. However, the development of both innate and acquired resistance to Menin inhibitors could limit their therapeutic potential. While studies have found that around 40% of these resistance cases can be traced back to mutations in the Menin gene, the Menin gene remains unaltered in other majorities, suggesting the presence of other mechanisms driving resistance. Here, we developed a unique chromatin-focused CRISPR knockout library, named Advanced Catalogue of Epigenetic Regulators (ACER). The ACER library contains approximately 800 traditional and newly-predicted epigenetic regulators using epigenetic network analyses that integrated publicly available gene codependency (DepMap) and protein-protein interaction data (BioPlex) (Figure A). To understand the chromatin mechanisms responsible for Menin-MLL inhibition resistance, we employed the ACER library for CRISPR screen in the context of Menin inhibition. Notably, our screen identified non-canonical polycomb repressive complex PRC1.1 (PCGF1, BCOR, and RYBP) and PRC2.2 (EZH2, SUZ12, and EED) among the top hits that conferred resistance to MLL-Menin inhibition upon gene depletion (Figure B). Consistent with our screen results, the depletion of PRC1.1 components led to a markedly increase in IC50 values when treated with the Menin inhibitor VTP50469 in both human and murine KMT2A-rearranged cell models. While treatment with Menin inhibitors typically induces cellular differentiation in leukemia cells, depletion of PRC1.1 genes resulted in an almost complete blockage in this differentiation process. Menin inhibitors evict Menin from chromatin and repress the expression levels of Menin-MLL target genes like MEIS1, PBX3, and MEF2C. Strikingly, our ChIP-seq and RNA-seq data indicatethat the loss of PRC1.1 components doesn't impact Menin's displacement from chromatin or the repression of KMT2A target genes by the Menin inhibitor. This is consistent with recent data from phase I clinical trial of revumenib (SNDX-5613) and preclinical patient derived xenograft models, wherein the Menin inhibitor persistently inhibitedkey KMT2A targets, even in resistant samples. These findings highlightthe possibility that resistance to Menin inhibition arising from mechanisms independent of canonical KMT2A target genes. Importantly, our unbiased transcriptomic analysis revealed that MYC signatures as the most affected pathway in PRC1.1-depleted cells following Menin inhibition. Furthermore, we show that both the transcriptionally active Menin-MLL and the repressive PRC1.1 complexes coexist at the MYC promoter, and the interplay between these two complexes determines MYC transcription output in AML. Moreover, enforced expression of MYC leads to resistance against Menin inhibition, while genetic and pharmacologic targeting MYC in PRC1.1 deficient cells rescued the resistance to Menin inhibition. Finally, in our RNA-seq datasets, we observed a diminished monocyte differentiation signature after PRC1.1 depletion. Considering AML cells with monocytic signatures, typically seen in KMT2A-rearranged AML, often exhibit resistance to BCL-2 inhibitor venetoclax, we hypothesized that AML cells lacking PRC1.1 could be more susceptible to BCL2 inhibition. This was confirmed in our ACER library screen with venetoclax treatment, where the PRC1.1 components PCGF1 and BCOR stood out as top epigenetic factors rendering drug sensitivity. Our subsequent studies using murine AML models and human primary samples further validated that PRC1.1-deficient AML cells are markedly sensitive to venetoclax. Furthermore, we show that venetoclax can be employed to overcome the resistance to Menin-MLL inhibition. In summary, our study identifiesthe non-canonical PRC1.1 as a key epigenetic driver of Menin-MLL resistance through a KMT2A target gene-independent mechanism which involves aberrant activation of MYC. We have further provided evidence that AML cells with loss of PRC1.1 are hypersensitive to BCL-2 inhibitor Venetoclax, opening a new therapeutic avenue for tackling Menin-resistant AMLs driven by polycomb inactivation.

Phase 1/2 First-in-Human Study of the Menin-MLL Inhibitor DSP-5336 in Patients with Relapsed or Refractory Acute Leukemia

Introduction: Menin inhibitors are an exciting new class of agents in development for patients (pts) with acute leukemia who overexpress HOXA9 and MEIS1 genes. Although there is a growing list of fusion genes in acute leukemia that appear to be dependent on homeobox-related gene activity, menin inhibition has been shown to be particularly active in pts with NPM1 mutations (NPM1c) and KMT2A rearrangements (MLLr). DSP-5336 is an investigational, oral small molecule designed to inhibit the menin and MLL protein interaction. Methods: A Phase 1/2 study of DSP-5336 is being conducted in pts with relapsed or refractory (R/R) acute leukemia. The dose escalation portion of the study consists of two parallel arms (Arm A: without concomitant anti-fungal azole therapy; Arm B: with concomitant azole therapy). Pts were eligible with R/R AML, ALL or acute leukemia of ambiguous lineage without a limit on number of prior therapies, with a focus on those with MLLr and NPM1c. Results: Accrual is ongoing with 24 pts enrolled as of April 2023; 14 pts in Arm A at doses of 40 mg BID (n=2), 80 mg BID (n=4), 100 mg BID (n=2) and 120 mg BID (n=6), and 10 pts in Arm B at doses of 40 mg BID (n=4) and 60 mg BID (n=6). The median age was 67.0 years (range 20 - 85) and 62.5% were male. All pts had AML except for 1 pt with acute leukemia of ambiguous lineage. Pts had received a median of 3 (range 1 to 9) prior treatments, and 6 (25%) had received prior alloSCT. MLLr or NPM1c were present in 6 (25%) and 4 (17%) patients, respectively. No dose-limiting toxicities have been observed thus far. Most treatment-emergent adverse events (TEAEs) were grade 1/2. TEAEs assessed as related to DSP-5336 in ≥ 3 pts were nausea (Grade 1 or 2, n = 3 pts) and vomiting (Grade 1 or 2, n = 4 pts). Grade >3 TEAEs occurring in ≥ 3 pts included anemia (6 pts), pneumonia (5 pts), sepsis (3 pts) and hypokalemia (3 pts), all unrelated to study drug. A possible Grade 4 differentiation syndrome (DS) was observed in 1 pt with a TP53 mutation in Arm A receiving 80 mg BID although concurrent pneumonia and sepsis made a clear attribution difficult. No other DS events of any grade have been reported to date. No cardiac toxicities, and no QTc prolongation of any grade related to DSP-5336 have been observed. Out of the 6 pts enrolled with MLLr, 4 were treated at dose levels projected to be active based on preclinical modeling (≥ 60 mg BID with azoles, or ≥ 120 mg BID without azoles). Of these 4 pts, all had received prior intensive induction chemotherapy as well as a venetoclax-based regimen, and 3 had received prior allo-transplant. Of these 4 pts, 1 achieved CRi with a duration of therapy of 5.1 months, 1 achieved MLFS with a duration of therapy of 6.2 months (ongoing), and 1 achieved SD with clearance of peripheral blasts, recovery of peripheral counts, resolution of leukemic gingival infiltration, and reduction in bone marrow (BM) blasts from 85% to 31%. Of the 4 enrolled pts with NPM1c, 2 were treated at doses projected to be effective. Stable disease with complete clearance of peripheral blasts were observed in both pts, and BM blasts were reduced by 66% and 83% respectively. Preliminary pharmacokinetic (PK) analysis has demonstrated generally higher mean exposures at higher dose levels. Across all the dose levels, plasma t 1/2 ranged from 2-6 hours in Arm A and from 2-10 hours in Arm B, with T max reached within 2 hours. Little to no drug accumulation was observed with repeat dosing in both arms. Data to date suggests that azoles may not have a significant effect on DSP-5336 exposure, but enrollment and comparative analyses are ongoing. Target pharmacodynamic changes have been achieved in pts who have MLLr or NPM1m, including rapid decreases in HOXA9, MEIS1, and PBX3 with treatment and, conversely, increases in CD11b compared to pretreatment. Conclusion: DSP-5336 has been well tolerated with no DLTs to date in heavily pretreated R/R AML patients with NPM1c and MLLr AML. Importantly, no cardiac signals (including no QTcF prolongation) have been observed. PK studies have not identified a significant drug-drug interaction with azoles. Although there are early signs of clinical activity and pharmacodynamic changes during administration of DSP-5336, the study is ongoing to determine a recommended Phase 2 dose for single-agent expansion and potential combination regimens. Updated safety and efficacy data will be presented at the meeting.

Early Results of the Phase I/II Study Investigating the All-Oral Combination of the Menin Inhibitor Revumenib (SNDX-5613) with Decitabine/Cedazuridine (ASTX727) and Venetoclax in Acute Myeloid Leukemia (SAVE)

Background: The interaction of menin with lysine methyltransferase 2A (KMT2A) is a dependency in acute leukemia caused by either rearrangement of the KMT2A (KMT2Ar) or Nucleoporin 98 ( NUP98r) genes, or mutation of the Nucleophosmin 1 gene ( NPM1mt). The menin inhibitor revumenib (previously SNDX-5613), is a potent, oral, selective inhibitor of the menin-KMT2A interaction with demonstrated safety and clinical activity in highly refractory acute leukemias (Issa GC, Nature 2023). KMT2Ar or NPM1mt leukemias are highly susceptible to induction of apoptosis through BCL2 inhibition, and dual Bcl-2 and menin inhibition led to synergistic activity in KMT2Ar or NPM1mt leukemia models (Carter BZ, Blood 2021). Therefore, we designed a phase I/II, investigator-initiated trial of the all-oral combination of revumenib, venetoclax and the hypomethylating agent ASTX727 in children and adults with relapsed/refractory (R/R) acute myeloid leukemia (AML) (NCT05360160). Methods: Patients (pts) with R/R AML or myeloid mixed-lineage acute leukemia (MPAL) aged 12 years and older were eligible.Dose escalation followed a 3+3 design. ASTX727 (decitabine/ cedazuridine) was administered at 35 mg/100 mg PO daily days 1-5, venetoclax at 400 mg (target dose) PO daily days 1-14, and revumenib 113 mg PO Q12h (dose level [DL] 0) or 163 mg PO Q12h (DL 1, used in phase II monotherapy), days 1-28 with either posaconazole or voriconazole (strong CYP3A4 inhibitors, for antifungal prophylaxis). Maintenance with revumenib monotherapy is planned following hematopoietic stem cell transplant (HSCT) for 1 year. For the first pt cohort, bone marrow examination was performed on cycle 1 day 14 and at the end of the cycle to assess for early morphologic remission and to inform future dose adjustments. Results: As of 7/20/2023, 8 pts were enrolled, 6 at DL0, and 2 at DL1. The median age was 27 years (range, 12-62 years), including 2 children (ages 12 and 16), 5 with KMT2Ar, 2 with NUP98r and 1 with NPM1mt. One pt had MPAL, and one had bone marrow and extramedullary disease. The median prior lines of therapy was 2.5 (range 1-4), 5 pts (63%) had prior venetoclax, 5 pts (63%) had a prior hypomethylating agent, 5 pts (63%) had prior HSCT, and one had prior menin inhibitor. The most common all-grade treatment-related adverse events (TRAEs) in ≥25% of pts were febrile neutropenia (63%), hyperphosphatemia (63%), nausea (63%), and AST/ALT elevation (25%). Grade ≥ 3 TRAEs were febrile neutropenia (63%), decreased platelets count (25%), and decreased neutrophil count (25%). There was 1 dose-limiting toxicity (DLT), grade 4 prolonged thrombocytopenia and neutropenia, at DL0 (resolved after dose hold), prompting enrollment of 3 additional pts, and escalation to DL1 when no additional DLTs were noted (1/6 pts with DLT). Pts at DL1 (N=2) have not completed the DLT period. There were no deaths due to TRAEs. No grade 3 or higher QTc prolongation occurred. Two pts had grade 2 differentiation syndrome (bone pain only), resolved with steroids. One pt ( NUP98r) had asymptomatic leukocytosis at the end of cycle 1 with neutrophilia, monocytosis, decrease in peripheral blasts, decrease in bone marrow blasts from 66% to 8%, and persistence of NUP98r by fluorescence in situ hybridization at 90% indicating differentiation. Seven of 8 pts are response-evaluable, and all 7 attained a morphologic remission (overall response rate of 100%) (Figure 1). Best responses achieved included a complete remission (CR) in 1 pt (including resolution of extramedullary disease), CR with partial hematologic recovery (CRh) in 1 pt, CR with incomplete platelet count recovery (CRp) in 3 pts, partial response (PR) in 1 pt, and 1 pt with morphologic leukemia free state (MLFS). Measurable residual disease was undetectable by flow (sensitivity at 10 -4) in 3 of 7 pts (43%). Three pts transitioned to HSCT following response, 2 are in continued remission, 1 has started maintenance and 1 pt died of transplant complications prior to starting maintenance. Enrollment is ongoing and updated data will be presented. Conclusions: Early results indicate acceptable safety and high efficacy of this combination in R/R myeloid leukemias with either KMT2Ar or NPM1mt or NUP98r. This study is ongoing with plans to establish the recommended phase 2 dose and optimize delivery of this combination.

Menin Maintains Cholesterol Content in Colorectal Cancer via Repression of LXR-Mediated Transcription.

Menin is a nuclear scaffold protein that regulates gene transcription in an oftentimes tissue-specific manner. Our previous work showed that menin is over-expressed in colorectal cancer (CRC); however, the full spectrum of menin function in colonic neoplasia remains unclear. Herein, we aimed to uncover novel menin-regulated pathways important for colorectal carcinogenesis. RNA-Seq analysis identified that menin regulates LXR-target gene expressions in CRC cell lines. Isolated colonic epithelium from Men1f/f;Vil1-Cre and Men1f/f mice was used to validate the results in vivo. Cholesterol content was quantified via an enzymatic assay. RNA-Seq analysis in the HT-29 CRC cell line identified that menin inhibition upregulated LXR-target genes, specifically ABCG1 and ABCA1, with protein products that promote cellular cholesterol efflux. Similar results were noted across other CRC cell lines and with different methods of menin inhibition. Consistent with ABCG1 and ABCA1 upregulation, and similarly to LXR agonists, menin inhibition reduced the total cellular cholesterol in both HT-29 and HCT-15 cells. To confirm the effects of menin inhibition in vivo, we assessed Men1f/f;Vil1-Cre mice lacking menin expression in the colonic epithelium. Men1f/f;Vil1-Cre mice were found to have no distinct baseline phenotype compared to control Men1f/f mice. However, similarly to CRC cell lines, Men1f/f;Vil1-Cre mice showed an upregulation of Abcg1 and a reduction in total cellular cholesterol. Promoting cholesterol efflux, either via menin inhibition or LXR activation, was found to synergistically suppress CRC cell growth under cholesterol-depleted conditions and when administered concomitantly with small molecule EGFR inhibitors. Menin represses the transcription of LXR-target genes, including ABCA1 and ABCG1 in the colonic epithelium and CRC. Menin inhibition conversely upregulates LXR-target genes and reduces total cellular cholesterol, demonstrating that menin inhibition may be an important mechanism for targeting cholesterol-dependent pathways in colorectal carcinogenesis.