MLL-rearranged AML Research Articles

The Clinical Menin Inhibitor Ziftomenib and the Nuclear Export Inhibitor Selinexor Synergistically Inhibit the Growth of MLL-r AML

CONCLUSION Menin is a scaffold protein that interacts with oncogenic histone-lysine-N-methyltransferase MLL1 (KMT2A)-fusion protein (FP) complex in MLL-rearranged ( MLL-r) AML. Menin inhibitors that evict menin from this interaction have been shown to be active against MLL-r and NPM1MT leukemia by modulating the expression of the leukemogenic homeobox A9 ( HOXA9) gene and its co-factor, MEIS1. Recent evidence showed that menin inhibitors can activate a tumor-suppressive network via a non-canonical transcriptional program through UTX. On the other hand, selective inhibitor of nuclear export (SINE) against XPO1/CRM1 has been shown to have robust anti-leukemia activity via enrichment of tumor suppressor proteins in the nucleus. In the present study, we hypothesized that menin and nuclear export inhibition would synergistically suppress AML cell proliferation and possibly sensitize menin inhibitor-resistant cells. We have used the clinical-stage menin inhibitor ziftomenib and SINE compound selinexor to simultaneously target menin-KMT2A protein-protein interactions and nuclear export. To assess growth inhibition, an ATP-based cell proliferation assay was used. Combination synergy was determined using CalcuSyn Version 2.0 synergy software. Stem-like progenitor cells were isolated using the StemSpan CD34+ expansion kit (StemCell Tech). Colony formation efficiency was determined using a Methocult assay (StemCell Tech). Gene and protein expression was detected using quantitative real-time PCR and western blotting. Flow cytometric analysis was performed to detect cell death and cell cycle status. We have performed the whole transcriptomic analysis via the RNA sequencing approach. The clinical candidate drug ziftomenib, in combination with selinexor, synergistically inhibited the growth of MLL-r AML cell lines (MV4;11 and MOLM13) (CI<1) [Fig.1A]. The combination of menin and XPO1 inhibitors significantly suppressed colony formation of CD34+ MLL-r progenitor stem cells derived from primary patient samples but was not toxic to normal CD34+ human hematopoietic progenitor stem cells (HPSCs). Menin inhibitor treatment caused the downregulation of menin protein along with its downstream targets HOXA9, MEIS1, and FLT3 in western blot analysis. Interestingly, selinexor also reduced menin, HOXA9, and MEIS1 protein levels. The combination enhanced menin down-expression, suggesting a molecular basis for the synergy between the two compounds. Ziftomenib suppressed the expression of menin in a time-dependent manner, which is evident as early as six hours of treatment. The combination showed increased CD11b, a marker of monocytic differentiation in MV4-11 treated cells. The combination also significantly enhanced both early and late apoptotic cell death in MLL-rearranged AML cells compared to either compound alone. Compared to controls, ziftomenib and selinexor-treated samples individually showed increased apoptotic and decreased G2/M populations, significantly enhanced in the combination. We observed differential expression of certain mRNAs and micro RNAs (miRs) in MV4-11 treated cells. The TMEM191B, SH3TC1, and SULT1C2 mRNAs were upregulated by ziftomenib and selinexor, which are enhanced further in the combination group. On the contrary, CENPK, CKS2, KIF18A, and H2BC4 mRNAs were downregulated by a single agent or combination. Among miRs, hsa-miR-34a-5p, hsa-miR-142-3p, hsa-miR-26b-5p were upregulated, and hsa-miR-33b-3p, hsa-miR-4454, hsa-miR-210-3p and hsa-miR-130b-3p [reported to be an essential lineage-specific codriver of MLL-AF4 leukemia] were downregulated by single agent or combination. Such mRNAs and miRs panels could serve as biomarkers for treatment response. MV4-11 cell line-derived xenografts in NSG mice showed a significant difference in survival in the combination arm compared to any single agent. Metronomic dosing also showed robust improvement in survival in the combination arm. Patient-derived xenografted NSG mice model using human primary MLLr re-transplantable leukemia cells showed improved survival in the combination arm [Fig. 1B]. These preclinical findings demonstrate that simultaneous inhibition of the menin-KMT2A interaction and nuclear export is a viable strategy for the treatment of MLL-r AML. Ongoing experiments with functional proteomic analysis will be presented at the ASH meeting.

Investigating the Role of the SETDB1 Target TANGO6 (TMCO7) in Leukemia

Despite advancements in AML therapies over the last 2 decades, 1 out of 3 patients diagnosed with AML succumb to their disease. The mainstay of treatment remains systemic chemotherapy, however better understanding of the genetic drivers in AML has generated new therapeutic opportunities. Targeted therapies have emerged to more precisely tailor leukemic treatment based on genetic and epigenetic drivers. We previously demonstrated that SETDB1, a H3K9 methyltransferase involved in heterochromatin regulation, negatively regulates the expression of proleukemic genes Hoxa9 and Meis1. Further, overexpression of SETDB1 correlates with improved overall AML patient survival and extends leukemic disease latency in mouse models (Ropa et al. Haematologica 2020). Here we describe a novel SETDB1 target, TANGO6, and aim to explore its role in leukemogenesis. We mined our genome wide expression and epigenetic datasets generated upon overexpression of SETDB1 to identify targets that may be important for leukemic progression (Ropa et al. Haematologica 2020). TANGO6 was found to be transcriptionally and epigenetically regulated by SETDB1, with little known about its role in leukemia. We performed a targeted CRISPR-Cas9 mediated knock out of TANGO6 to investigate its importance in several human leukemic cell lines: MV4;11, K562 and KOPN8. We established the above cell lines with Cas9 constitutively expressed and introduced sgRNAs targeting TANGO6 by lentivirus. We then performed a competitive growth assay using flow cytometry to assess cell fitness on days 3, 14, 10, 21. We observed that loss of TANGO6 resulted in decreased fitness across all three leukemic cell lines compared to the negative control. This phenotype was more striking in the K562 and KOPN8 cell lines. In lieu of having a reliable antibody to visualize changes in protein expression, we assessed mutational changes in the DNA sequence to evaluate efficiency of our CRISPR/Cas9 system. Genomic DNA was prepared from sg TANGO6 transduced K562 cells on days 5 and 10. The targeted region of TANGO6 was PCR amplified and TOPO cloned for direct Sanger sequencing. 92% (12 out of 13 samples) showed a mutation involving the target sequence. Of these mutations, ~67% resulted in frameshift mutations leading to early truncation of the protein. The remaining mutations involved deletions ranging from 1 to 28 amino acids, with one sample resulting in two single point mutations conferring a change of leucine to serine and lysine to arginine. These mutations may or may not affect the folding of the protein. This data demonstrates we are successfully targeting TANGO6, which produces the biological phenotype in our competitive growth assay. To further understand the potential importance of TANGO6 in human leukemia, we performed qPCR on 5 leukemic cell lines (2 MLL-rearranged AML cell lines, 2 B-lymphoblastic cell lines, and 1 CML cell line). Our data showed that TANGO6 was expressed at higher levels across all leukemic cell lines compared to normal CD34+ hematopoietic stem/progenitor cells, consistent with a role in leukemogenesis. Our preliminary data suggests that TANGO6 plays an important role in leukemogenesis. Ongoing work focuses on validating this hypothesis using in vivo leukemic mouse models and protein localization studies. Given the lack of available information on human TANGO6, characterizing its location and mechanism is essential to understanding its role in disease. Our work has established the need for further exploration of TANGO6 as a potential therapeutic target for AML.

Targeting HDAC9 Contributes to Degradation of MLL Fusion Oncoproteins in KMT2A-Rearranged Acute Myeloid Leukemia

Acute myeloid leukemia with MLL rearrangement (MLLr-AML) is a highly aggressive subtype of AML. Existing treatment strategies are nonspecific and ineffective. The MLL fusion gene is one of the driver oncogenes in leukemia and is central to the pathogenesis of MLLr-AML, and more research has recently begun to focus on targeting MLL fusion proteins. Targeting MLL fusion proteins has been shown to have a tumor suppressor effect, demonstrating efficacy in preclinical models of MLLr-AML, and in 2022 Scott A Armstrong ‘s team suggested that degradation of MLL-AF9 can cause an increase in cell differentiation and apoptosis, confirming the efficacy of targeting MLL fusions. However, specific targets that degrade MLL fusion proteins as well as clinically translatable targeted therapies are still lacking and need to be further explored. Study confirmed that MLL-rearranged ALL cells are highly sensitive to the broad-spectrum HDAC inhibitor panobinostat. A recent clinical study by Mireia Camós’ team proposed that HDAC9 has a significantly high expression in childhood MLL-rearranged B-cell precursor acute lymphoblastic leukemia (BCP-ALL) and AML, but that article did not address any mechanistic studies. The potential value of HDAC9 in adult lymphoid hematologic diseases has been gradually revealed, however, the expression profile and precise molecular mechanisms of HDAC9 in adult myeloid hematologic tumors, have not been reported, which deserves to be further explored. In order to investigate the expression and role of HDAC9 in MLLr-AML, we analyzed the second-generation sequencing data from the Beat AML dataset and found that HDAC9 has a significantly high expression in MLLr-AML, while only HDAC9 is characterized by this feature in HDAC family molecules. Further we analyzed the data of 300 adult AML specimens in our hospital (including 20 MLLr-AML patients), and the results further confirmed that only HDAC9 among the HDAC family had a significantly higher expression level in the MLLr-AML patient group than that in the Non-MLLr-AML patient group (P<0.001), and the high expression of HDAC9 in the MLLr-AML patient group was a common phenomenon and not limited to a particular class of MLL chaperone molecules. Further studies revealed that overexpression of MLL fusion proteins in HEK293T could cause up-regulation of HDAC9 protein levels, and this experiment clarified the direct correlation between the MLL fusion genes and the high expression of HDAC9. The HDAC IIa inhibitor (TMP-269), which can target HDAC9, was significantly more sensitive in MLLr-AML cell lines than in Non-MLLr-AML cell lines, and also that MLLr-AML cell survival was dependent on HDAC9, and that knockdown of HDAC9 induced an increase in apoptosis and a slowdown in proliferation in MLLr-AML cells. We subsequently demonstrated that knockdown of HDAC9 downregulates the expression of MLL fusion proteins through degradation of MLL proteins. Furthermore, when exploring therapeutic options for targeting MLLr-AML, we found that combining the BCL-2 inhibitor Venetoclax (VEN) with an MLL-Menin specific inhibitor (MEN1i) specifically downregulated the expression of HDAC9 and had a favorable synergistic killing effect on MLLr-AML in both in vitro and in vivo models, further suggesting an important role of HDAC9 in the treatment of MLLr-AML. To sum up, through this study, we can shed light on the role of specific HDAC molecules in MLLr-AML and provide a new potential target for the degradation of MLL fusion protein to eradicate MLLr-AML.

Molecular Relapse in Pediatric AML: Clinical Relapse May be Predicted By Increasingin MRD Level - Experiences from Real-World Data in Patients with Inv(16), t(8;21), t(9;11), t(10;11) or NPM1

Introduction Detection of molecular relapses may allow early intervention aiming improved survival or , at least, reduced toxicities. However, so far the impact of molecular relapses in pediatric AML is not yet determined. Therefor this retrospective analysis outlines the prediction of clinical relapses following molecular relapses in pediatric patients inv(16), t(8;21), t(9;11), t(10;11) or NPM1. Patients and Methods Patients included in this analysis was initially diagnosed with an AML according to WHO with at least one quantitative marker, availability of an initial sample (day 0) and more than three follow up measurements until 18 months (d=532) after date of diagnosis. Between 2015 and 2020 n=258 children with inv(16) (n=52; 20%), t(8;21) (n=84; 32%), t(9;11) (n=64;25%), t(10;11) (n=20; 8%) and NPM1 (n=38; 15%) underwent MRD monitoring by rt-PCR in bone marrow (BM). Monitoring in peripheral blood (PB) was also recommended every 4 weeks. In all patients (molecular) CR must be confirmed at the start of last consolidation. Based on the marker specific sensitivity a molecular relapse was defined as an increase in MRD level by at least 1 log to a level greater than the marker specific baseline, with a normal percentage (<5%) of myeloblasts in BM and the absence of proven histological extramedullary relapse. Patients were censored if they had a clinical relapse or reach the age of 18 years. Results For inv(16) 60% (n=32), t(8;21) 82% (n=69), t(9;11) 17% (n=17), t(10;11) 60% (n=12) and NPM1 39% (n=15) of the patients meet the inclusion criteria. In inv(16) patients, n=1 (3%) molecular relapse could be detected, which was followed within 4 weeks by a clinical relapse. One clinical relapse was recorded 146 days after an increasing signal in PB, but without a corresponding molecular relapse. In the t(8;21) cohort, n=7 patients was diagnosed with a molecular relapse, 4/7 were treated pre-emptive with azacytidine. 5 patients got a clinical relapse afterwards, n=2 did not relapse open until the end of the observational timeframe to d532. In n=10 patients the clinical relapse could not be predicted by rt-PCR. For MLL-rearranged AML such as t(9;11) and t(10;11) molecular relapses was diagnosed in n=5 (45%) and n=2 (17%) patients. None of them was pre-emptively treated. In 60% (n=3) for t(9;11) and 100% (n=2) for t(10;11) clinical relapses followed. Interestingly, that just one patient (17%) with t(9;11) had a clinical relapse without a prior molecular relapse, but in 40% (n=10) of the t(10;11) group, the clinical relapse was not molecularly predictable. In n=2 of the molecular relapses in t(9;11) patients no clinical relapse followed within the observation period. In NPM1 positive patients n=2 molecular relapses (13%) were diagnosed, both were treated with azacitidine, both relapsed open. In this group was no patient with a clinical relapse without a prior molecular relapse. Conclusion In all groups MRD level measured by rt-PCR could be an effective diagnostical tool in pediatric AML to detect molecular relapses prior to clinical relapses opening a window for pre-emptive therapy. Obviously in MLL-rearranged AML like t(9;11) or t(10;11) kinetics seems to be too fast (at least for an epigenetic intervention) that, once the molecular relapse is detected, clinical relapse follows immediately or, the MRD levels increase so rapidly, that the molecular relapse cannot be detected within the recommended analysis interval of 4 weeks. Same seems to be apply to patients with NPM1. In core-binding leukemia such as inv(16) and t(8;21) the kinetics leads to enough time to step into the pre-emptive therapy with azacitidine. Here is predictability pending on a close monitoring in PB and BM, which needs to be implemented without time gaps in sampling in everyday clinical practice to keep this additional treatment chance for patients. Even if exemptions are very rare (3%) and can be most likely explained by errors during lab measurement, molecular relapses in pediatric patients with inv(16), t(8;21), t(9;11), t(10;11) or NPM1 leads to clinical relapses over time. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Novel Combination of Clinical Menin Inhibitor Ziftomenib and the Nuclear Export Inhibitor Selinexor Synergistically Inhibit MLL-r AML

PURPOSE: Menin is a scaffold protein that tethers histone-lysine-N-methyltransferase MLL1 (KMT2A) to chromatin by binding to the menin binding domain (MBD) of KMT2A. The N-terminus of the KMT2A gene recombines with a range of genes to form more than 80 fusion proteins (FP) in MLL-rearranged (MLL-r) AML. In myeloid stem progenitor cells, the menin-KMT2A complex modulates the expression of the leukemogenic homeobox A9 (HOXA9) gene, as well as its co-factor, MEIS1. Menin inhibitors evict menin from the chromatin and reduce KMT2A and KMT2A-FP binding to their targets in MLL-r leukemia, breaking the differentiation block and inducing tumor regression. Aberrant nuclear export is common in cancer, resulting in anomalous localization of various proteins, including tumor suppressor proteins (TSPs). Blocking XPO1/CRM1 using a selective inhibitor of nuclear export (SINE) compound selinexor has been shown to have robust anti-leukemia activity. In the present study, we hypothesized that inhibition of menin and nuclear export would synergistically suppress AML cell proliferation and survival. We have used the clinical-stage menin inhibitor ziftomenib and selinexor to simultaneously target menin-KMT2A protein-protein interactions and nuclear export. METHODS: To assess growth inhibition, an ATP-based cell proliferation assay was used. Combination synergy was determined using CalcuSyn Version 2.0 synergy software. Stem-like progenitor cells were isolated using the StemSpan CD34+ expansion kit (StemCell Tech). Colony formation efficiency was determined using a Methocult assay (StemCell Tech). Gene and protein expression and cell death were detected using quantitative real-time PCR, western blotting, and annexin-V/PI fluorescence-activated cell sorting, respectively. RESULTS: The clinical candidate ziftomenib, in combination with selinexor, synergistically inhibited the growth of MLL-r AML cell lines (MV4;11, MOLM13, and SEM) (CI<1) [Fig.1A]. The combination of menin and XPO1 inhibitors significantly suppressed colony formation of CD34+ MLL-r progenitor cells derived from primary patient samples but was not toxic to normal CD34+ human hematopoietic progenitor stem cells (HPSCs). Menin inhibitor treatment caused the downregulation of menin protein along with its downstream targets HOXA9, MEIS1, and FLT3 in western blot analysis. Interestingly, the nuclear export inhibitor selinexor also reduced menin, HOXA9, and MEIS1 protein levels. The combination of menin and XPO1 inhibitors enhanced menin down expression, suggesting a molecular basis for the synergy between the two compounds [Fig 1.B]. Ziftomenib suppressed the expression of menin in a time-dependent manner, which is evident as early as six hours of treatment. This combination significantly enhanced both early and late apoptotic cell death in MLL-rearranged AML cells compared to either compound alone. We have performed total RNA sequencing and observed differential expression of certain mRNAs and micro RNAs (miRs). The TMEM191B, SH3TC1, and SULT1C2 mRNAs were upregulated by KO539 and Selinexor, which are enhanced further by combination treatment. On the contrary, CENPK, CKS2, KIF18A, and H2BC4 mRNAs were downregulated by a single agent or combination. Among miRs, hsa-miR-34a-5p, hsa-miR-142-3p, hsa-miR-26b-5p were upregulated and hsa-miR-33b-3p, hsa-miR-4454, hsa-miR-210-3p and hsa-miR-130b-3p [reported to be an essential lineage-specific codriver of MLL-AF4 leukemia] were downregulated by single agent or combination. Such mRNAs and miRs panels could serve as biomarkers for treatment response. CONCLUSION: These preclinical findings demonstrate that simultaneous inhibition of the menin-KMT2A interaction and nuclear export could be a viable strategy for the treatment of MLL-r AML. Ongoing experiments with CDX and PDX using MLL-r models and functional analysis using total transcriptome sequencing and proteomics will be presented at the ASH meeting. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Identification of a Novel FAM135B Cancer Susceptibility Mutation in a Family with Multiple Cases of MLL-Rearranged AML

Background: Familial aggregation of malignancies, including leukemia, is rarely reported due to the scattered cases, weakened bonding among generations, and low birth rates in modern society. The emerging demands for predicting and preventing cancer development are outgrowing the progress in the studies of the mechanism underlying the propensity of malignancies. Results: Here we reported a family with 4 cases of acute leukemia (AL) and four solid tumors (2 for lung cancer, 1 for renal clear cell carcinoma, and one for rectal adenocarcinoma). Besides, one member in the family was diagnosed with intraspinal cavernous hemangioma, and another one was diagnosed with congenital heart disease (transposition of the great arteries). 2 out of 4 AL patients were cousins and diagnosed with acute monocytic leukemia (AML-M5 with MLL-rearrangement) in our center. Whole genome sequencing was performed on two patients with AML, two patients with solid tumors, one obligated carrier, and three healthy members. Using an in-house bioinformatic data analysis pipeline, we identified 18 single nucleotide variants (SNVs) and four insertions/deletions (Indel) in all the affected members and one obligated carrier while absent in healthy members. Genotyping of these variants in 57 family members confirmed that 18/22 variants co-segregated with the malignant phenotype of this family. By screening for these 18 variants in 13 AL pedigrees, we narrow our cancer-predisposing candidate list to one variant, hitting FAM135B (family with sequence similarity 135 member B; NM_015912: exon13:c.A2621G:p.E874G), which is recurrent in another AML pedigree with two cases in two consecutive generations. This mutation resides in a highly conserved locus among species and is predicted to be deleterious. FAM135B is predicted to encode serine esterase, and the role of FAM135B in AML was not documented. Intriguingly, all the affected familial AML cases from two pedigrees were diagnosed with AML-M5 with MLL-rearrangement. Moreover, this mutation is recurrent in sporadic adult AML cohort (6/467), among which half (3/6) were AML-M5, two M3, and one M2. Functionally, we found that overexpression of FAM135B promotes the proliferation and colony formation of AML cells. Consistently, downregulation of FAM135B nearly abolished the colony-forming capacity of AML cells and impaired cell proliferation. Further studies on the role of FAM135B in AML development are under investigation. Conclusion: We identify a novel cancer-predisposing gene, FAM135B, and its mutation E874G occurred in both familial and sporadic AML cases, especially AML-M5 with MLL-rearrangement.

Clinical-Stage Menin Inhibitor KO-539 Is Synergistically Active with Multiple Classes of Targeted Agents in KMT2A-r and NPM1-Mutant AML Models

The N-terminus of the histone-lysine-N-methyltransferase MLL1 (KMT2A) contains the menin binding domain (MBD). Menin is a scaffold protein that binds to MBD and tethers MLL1 to chromatin. The Menin-KMT2A complex regulates expression of the leukemogenic homeobox A9 (HOXA9) gene and its co-factor MEIS1 in myeloid stem progenitor cells. In MLL-rearranged (MLL-r) AML, the N-terminus of the KMT2A gene is fused to the C-terminus of any of over 80 fusion partners, most commonly AF4, AF9, ENL and ELL. These MLL fusion partners are components of (and recruit) the super elongation complex and DOT1L to induce H3K4Me3 and H3K79Me2 marks on active chromatin, driving aberrant expression of HOXA9, MEIS1, PBX3, MEF2C and CDK6. In AML with mutant NPM1 (NPM1c), the wild type menin-KMT2A complex is the main oncogenic driver of HOXA9, MEIS1 and FLT3, promoting self-renewal of mutant leukemic blasts. KO-539 is a novel, oral investigational drug candidate targeting the Menin-KMT2A protein-protein interaction. Preclinical data show that KO-539 and close analogs induce differentiation and loss of viability of AML cells, and exert profound anti-leukemic activity in multiple PDX models harboring MLL-FP or NPM1c when dosed continuously QD for 3-6 weeks. In an ongoing phase 1/2A KOMET-001 clinical trial (NCT04067336) evaluating KO-539 in adult patients with relapsed and/or refractory AML, significant biological activity as well as complete remission lacking minimal residual disease have been observed.The present studies were focused on further elucidating biologic effects and on determining synergistic activity of KO-539 with agents targeting BCL2, BET proteins and CDK6. Treatment with KO-539 dose-dependently inhibited in vitro growth, as well as induced differentiation (by both morphology and increased CD11b expression) and loss of viability of MOLM13 (MLL-AF9 and FLT3-ITD) and OCI-AML3 (NPM1c and homozygous NRAS mutation) cells and patient-derived (PD) MLL-r or NPM1c AML cells. This was associated with repression of MEIS1, PBX3, MEF2C, FLT3, MYC, BCL2 and CDK6 transcription but increased mRNA expression of ITGAM (CD11b). KO-539 treatment also dose-dependently depleted protein levels of Menin, MEIS1, FLT3, CDK6 and BCL2, but upregulated MCL1 and CD11b proteins in MOLM13 and OCI-AML3 cells. Mass cytometry (CyTOF) analysis of PD MLL-r and NPM1c AML samples confirmed that, following KO-539 treatment there was also decline in protein levels of Menin, MEIS1, MEF2C, PBX3, FLT3, CDK6 and BCL2 in phenotypically characterized AML stem cells (with high expression of CLEC12A, CD123, CD244, CD99, but low expression of CD11b). KO-539-mediated Menin depletion was associated with polyubiquitylation and proteasomal degradation of Menin protein, given that protein levels were restored by co-treatment with the proteasome inhibitor carfilzomib. Notably, co-treatment (in vitro) with KO-539 in combination with venetoclax, OTX015 (pan-BET inhibitor) or abemaciclib (CDK6 inhibitor) for 72 to 96 hours induced synergistic loss of viability in cultured cell lines and PD AML cells from both MLL-r and NPM1c AML but not normal CD34+ progenitor cells or AML cells lacking MLL-FP or NPM1c, as determined by the SynergyFinder algorithm. Findings of ongoing in vivo studies determining effects of treatment with KO-539 and/or venetoclax or OTX015, versus vehicle control in PDX models will be presented at the meeting. These preclinical findings highlight the molecular correlates of anti-AML efficacy of KO-539 and demonstrate potentially synergistic KO-539-based combinations with inhibitors of BCL2, BET proteins and CDK6 against MLL-r or NPM1 mutant AML. DisclosuresDaver: Bristol Myers Squibb: Consultancy, Research Funding; Astellas: Consultancy, Research Funding; Abbvie: Consultancy, Research Funding; Trovagene: Consultancy, Research Funding; Gilead Sciences, Inc.: Consultancy, Research Funding; Novimmune: Research Funding; FATE Therapeutics: Research Funding; Genentech: Consultancy, Research Funding; Glycomimetics: Research Funding; Amgen: Consultancy, Research Funding; Hanmi: Research Funding; Sevier: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Trillium: Consultancy, Research Funding; ImmunoGen: Consultancy, Research Funding; Novartis: Consultancy; Jazz Pharmaceuticals: Consultancy, Other: Data Monitoring Committee member; Dava Oncology (Arog): Consultancy; Celgene: Consultancy; Syndax: Consultancy; Shattuck Labs: Consultancy; Agios: Consultancy; Kite Pharmaceuticals: Consultancy; SOBI: Consultancy; STAR Therapeutics: Consultancy; Karyopharm: Research Funding; Newave: Research Funding. Kadia: Aglos: Consultancy; AbbVie: Consultancy, Other: Grant/research support; Amgen: Other: Grant/research support; BMS: Other: Grant/research support; Cure: Speakers Bureau; Genentech: Consultancy, Other: Grant/research support; Dalichi Sankyo: Consultancy; AstraZeneca: Other; Astellas: Other; Genfleet: Other; Ascentage: Other; Cellonkos: Other; Sanofi-Aventis: Consultancy; Pulmotech: Other; Jazz: Consultancy; Liberum: Consultancy; Novartis: Consultancy; Pfizer: Consultancy, Other. DiNardo: Forma: Honoraria, Research Funding; Foghorn: Honoraria, Research Funding; Agios/Servier: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Research Funding; Notable Labs: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria; Novartis: Honoraria; GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Research Funding; ImmuneOnc: Honoraria, Research Funding; Celgene, a Bristol Myers Squibb company: Honoraria, Research Funding. Leoni: Kura Oncology: Current Employment. Burrows: Kura Oncology: Current Employment.

Safety and Efficacy of Menin Inhibition in Patients (Pts) with MLL-Rearranged and NPM1 Mutant Acute Leukemia: A Phase (Ph) 1, First-in-Human Study of SNDX-5613 (AUGMENT 101)

Safety and Efficacy of Menin Inhibition in Patients (Pts) with MLL-Rearranged and NPM1 Mutant Acute Leukemia: A Phase (Ph) 1, First-in-Human Study of SNDX-5613 (AUGMENT 101)

Inhibition of Oxphos with Iacs-010759 Depletes AML Leukemia Initiating Cells (LIC) in Vitro and Improves Survival in Pre-Clinical AML Models

Introduction: Metabolic reprogramming of energy-generating pathways is a core oncogenic property of leukemias and other cancers that provides a potential therapeutic vulnerability.In light of recent studies demonstrating reliance of Leukemia Initiating Cells (LIC) on OxPhos (Skrtic et al., 2011; Lagadinou at al., 2013), we aimed to determine the effects of OxPhosi on AML bulk and LICs in vitro and in vivo using patient-derived xenograft (PDX) AML models.Methods and Results: Our gene set enrichment analysis conducted on publicly available gene expression profiles of LIC-enriched populations (Ng et al., 2016) confirmed significant enrichment of OxPhos-related genes relative to no LIC. Using transmission electron microscopy (TEM), we found a significant increase in mitochondria numbers in 8 primary CD34+ AML samples compared with 3 healthy bone marrow (hBM) CD34+ sample (19±8 vs 8±1,p=0.01, Fig. 1A), consistent with previously reported data (Škrtić at al.,2011). This finding provides rationale for assessing OxPhos as a potential target for therapeutic intervention.IACS-010759 is a novel potent inhibitor of OxPhos (OxPhosi) that blocks cellular respiration through inhibition of mitochondrial complex I (Molina et al., AACR2016).In 80% of the AML samples studied (n=50), IACS-010759 exposure induced profound growth-inhibitory effects in vitro at an EC50 of 3.1nM (0.1-11.7nM) and 5.0 nM (0.9-12.8nM) in AML blasts and CD34+ AML LIC respectively, with minimal toxicity against hBM CD34+ cells. In line with viability data, OxPhosi reduced colony forming ability of LIC (n=5, EC50 13-30nM) but not of hBM CD34+ cells. Further, IACS-010759 induces differentiation of CD34+ cells in a subset of AML samples as determined by flow cytometric analysis of colonies collected after treatment. Next, profound reduction of both, oxygen-dependent ATP production and spare respiratory capacity (SRC), at IC50s of 20.5 nM (range, 1.3*10-5 -528) for bulk and 51.4 nM (range, 1.3-528) for CD34+ cells, was shown by Seahorse assay. This translated into the mitochondrial membrane potential loss (TMRE, JC1 flow cytometry), mitochondria swelling and degeneration shown by TEM.Based on genomic characteristics and responses to OxPhosi in vitro, two distinct groups of patients (pts) with unique metabolic pattern based on SRC were identified: samples from pts with low SRC (36.3±17.4 pmol/min) enriched in IDH1/2-mutated AML were sensitive to IACS-010759 with median 60% of viability drop at 123 nM, while samples with high SRC (86.7±20), enriched in TP53-mutated AML were resistant to OxPhosi with 30% viability reduction (p=0.03).The proteome studies of AML pts samples revealed dose-dependent increased expression of energy stress response protein pAMPK, upregulation of glycolysis marker PFKFB and decrease of the fatty acids oxidation markers CPT1 and ACC1.Further, the intracellular metabolome analysis of LIC from 4 primary AML pts showed modulation of amino acids (AA) with increased level of glutamine and aspartate, decreased TCA intermediates and increased lactate level indicative of OxPhos perturbation. In contrast, only moderate decrease of AA level and TCA intermediates was observed in hBM cells, consistent with lack of cytotoxicity on hBM. In a PDX model from MLL-rearranged AML refractory to standard chemotherapy, IACS-010759 treatment prolonged survival and delayed disease progression (Molina et al., AACR 2016). Mass cytometry analysis of BM and spleen cells demonstrated decrease of LIC (CD34+, CD38-, CD123+, CD33+), relative increase of CD11c+ differentiated cells and suppression of proliferation measured by Ki67 (Fig. 1B). The depletion of LIC by OxPhosi was confirmed in a secondary transplantation experiment. Mice injected with similar numbers of BM AML cells isolated from primary engrafted recipients of vehicle, Ara-C or IACS-010759 , showed improvement of the overall survival in OxPhosi arm (Fig. 1C).Conclusion: Our study demonstrates that inhibiting OxPhos with IACS-010759 constitutes a novel promising therapeutic approach that targets a unique metabolic vulnerability of AML LICs via energy depletion, inhibition of proliferation and induction of differentiation. We have shown for the first time that IDH1/2 mutated AML have low spared respiratory capacity and are highly sensitive to IACS-010759. These findings are being tested in an ongoing Phase 1 clinical trial of IACS-010759 in AML (NCT02882321). [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

MiR-181a enhances drug sensitivity of mixed lineage leukemia-rearranged acute myeloid leukemia by increasing poly(ADP-ribose) polymerase1 acetylation

Chromosomal translocations and rearrangements involving Mixed Lineage Leukemia (MLL) gene is associated with poor prognosis in AML. Extensive epigenetic changes were found in this group of patients. In clinical study, we found miR-181a expression level was significantly lower in MLL-rearranged AML. As an important epi-miRNA, the role of miR-181a as an epigenetic regulator in leukemia has not been investigated before. In this study, we found miR-181a overexpression enhanced total protein acetylation in THP-1 cells, which harbor MLL-AF9 fusion gene, and protein Mass Spectrum identified poly(ADP-ribose) polymerase 1 (PARP1) was a major downstream target. Increased PARP1 acetylation was mediated by down-regulation of histone deacetylase Sirtuin1 (Sirt1). MiR-181a overexpression resulted in DNA trapping of PARP1, increased DNA double strand break formation and increased chemosensitivity of leukemia cells both in vitro and in vivo. This study indicates miR-181a-Sirt1-PARP1 acetylation pathway could be a promising target for this special group of AML.

Clinical Significance of Common Gene Mutations in 53 Patients with Acute Myeloid Leukemia Harboring 11q23/MLL Rearrangements

To investigate the clinical significance of AML patients with 11q23/MLL rearrangement, and to evaluate the effect of those mutations on the AML patients. 53 cases involving translocations of chromosome 11q23 were identified by chromosome banding analysis. MLL rearrangements were detected by fluorescence in situ hybridization and/or multiplex nested PCR. The samples were screened for mutations in the candidate genes FLT3-ITD, FLT3-TKD, TET2, N-RAS, ASXLI, EZH2, DNMT3, C-Kit, NPM1, WT1, CEBPA by using genomic DNA-PCR and deep-sequencing. 21/53 MLL-rearranged AML cases showed at least one additional chromosomal aberrations. The most common additional aberration was +8. Gene mutations were observed in 23 cases (43.4%) and most cases showed singal mutation. N-RAS mutation was more frequent (8 cases, 15.1%), followed by WT1 mutation in 4 cases (7.5%), FLT3-ITD mutation in 3 cases, ASXL1 mutation in 2 cases, DNMT3A mutation in 2 cases, EZH2 mutation in 1 case, c-Kit17 mutation in 1 case, FLT3-TKD mutation in 1 case, and FLT3-ITD and TKD mutation coexistent in 1 case. No mutation was detected in CEBPA, NPM1, C-KIT8, TET2. Median OS for gene mutated patients was 8.5 months and 13 months for no mutated patients. Median OS for patients who received hematopoietic stem cell transplantation (HSCT) was 22.5 months and 7.5 months for patients who olny received chemotherapy. A relatively high mutation frequency is observed in AML patients with 11q23/MLL rearrangements and most cases shows single mutation. The RAS signaling pathway alterations are most common. Gene mutation does not affect the OS of these patients, who show poor prognosis. A significantly higher Hb at initial diagnosis in FLT3 mutated patients is significantly higher than that in FLT3 wild-type cases. Patients who underwent HSCT show a better prognosis than those only received chemotherapy.

Aberrant TRPM4 expression in MLL-rearranged acute myeloid leukemia and its blockade induces cell cycle arrest via AKT/GLI1/Cyclin D1 pathway

Transient Receptor Potential Melastatin Subfamily Member 4 (TRPM4) has been demonstrated to be aberrantly expressed in several cancers but seldom reported in acute leukemia. Based on database mining and validated experiments, our present data show that TRPM4 is selectively overexpressed in AML patients and cell lines with the MLL gene rearrangement. We analyzed the correlation between TRPM4 expression and clinical parameters in a validated cohort of AML patients. Increased TRPM4 expression was associated with significant leukocytosis (p = .028), M4/M5 subtype (p = .000), FLT3-ITD mutation (p = .034), MLL status (p = .007) and a higher risk stratification (p = .001). Knockdown of TRPM4 mediated by siRNA impaired proliferation and arrested the cell cycle at the G0/G1 phase in MLL-rearranged leukemia cells. We suggested that TRPM4 may be involved in the pathogenesis of MLL-rearranged leukemia through regulating the AKT/GLI1/Cyclin D1 pathway. The transcription factor HOXA9 was found to be responsible for upregulation of TRPM4 expression by binding to its promoter. In conclusion, TRPM4 is overexpressed in MLL-rearranged AML and blockade of TRPM4 may be an alternative therapeutic approach in AML patients with high TRPM4 expression.

FOXM1 regulates leukemia stem cell quiescence and survival in MLL-rearranged AML

FOXM1, a known transcription factor, promotes cell proliferation in a variety of cancer cells. Here we show that Foxm1 is required for survival, quiescence and self-renewal of MLL-AF9 (MA9)-transformed leukemia stem cells (LSCs) in vivo. Mechanistically, Foxm1 upregulation activates the Wnt/β-catenin signaling pathways by directly binding to β-catenin and stabilizing β-catenin protein through inhibiting its degradation, thereby preserving LSC quiescence, and promoting LSC self-renewal in MLL-rearranged AML. More importantly, inhibition of FOXM1 markedly suppresses leukemogenic potential and induces apoptosis of primary LSCs from MLL-rearranged AML patients in vitro and in vivo in xenograft mice. Thus, our study shows a critical role and mechanisms of Foxm1 in MA9-LSCs, and indicates that FOXM1 is a potential therapeutic target for selectively eliminating LSCs in MLL-rearranged AML.

MLL-Rearranged AML Is Associated with Poor Outcomes As Compared to Patients with Intermediate- and Adverse-Risk Disease: A CIBMTR Study of 3779 Adult Patients

Background Studies evaluating the impact of 11q23 / KMT2A / MLL translocations on post-transplant outcomes as compared to other intermediate- and adverse-risk cytogenetic characteristics are lacking. Methods We considered 8709 AML patients from the CIBMTR database for inclusion. We evaluated relapse, non-relapse mortality (NRM), disease-free survival (DFS), and overall survival (OS) for patients who were transplanted in CR1 stratified by presence of 11q23 rearrangement, intermediate- or adverse-risk disease. Assignment of cytogenetic risk was performed by hand, based on the 2010 AML-MRC Classification System. A multivariate analysis was performed using a Cox proportional hazards model. Results Characteristics of 3779 patients, selected based on disease and donor type, HSCT in CR1, and availability of data, are shown in Figure 1. Of these, 426 patients had an 11q23-rearrangement, 2384 had intermediate-risk disease, and 969 had adverse-risk disease. Of the patients with 11q23 translocations, 112 (26%) had t(9;11), 62 (15%) had t(11;19), 41 (10%) had t(6;11), 28 (7%) had t(10;11), and 47 (11%) had other partners. No partner was provided in 136 cases (32%). Figures 2 and 3 show the DFS, OS, NRM, and relapse data stratified by cytogenetic category. Based on the multivariate analysis, DFS was lowest in adverse-risk patients (HR for death 1.47, 1.26 for MLL, p Stratification of outcomes by translocation partner did not yield any statistically significant differences between groups, however a descriptive analysis of these data will be presented. Measurable residual disease (MRD) status did not appear to play an independent role in NRM or in OS. However, the multivariate HR for relapse in patients with MRD-positivity was 1.23, p Conclusions Patients with MLL-rearranged AML had post-HSCT DFS and OS that were more similar to patients with adverse-risk disease than to those with intermediate-risk characteristics. MLL patients had a higher probability of relapse than intermediate-, but not adverse-risk, patients. NRM was similar among all groups. Both prognostic classification and MRD status independently contributed to the risk of relapse and DFS, but MRD was not an independent factor in predicting NRM or OS. Our study suggests that patients with MLL rearrangements do almost as poorly as those with adverse-risk disease and that novel approaches for this high-risk population represent an urgent unmet clinical need.

Hottip Lncrna Reinforces CTCF Defined Chromatin Boundaries and Drives Wnt Target Gene Expression in AML Leukemogenesis

We reported recently that HOXA locus associated lncRNA, HOTTIP, is highly expressed in AML patients carrying MLL rearrangement and NPM1c+ mutations. The expression of HOTTIP positively correlates with posterior HOXA gene expression and poor patient survival. We further demonstrated that HOTTIP acts as an epigenetic regulator to define oncogenic HOXA topologically associated domain (TAD) and drive HOXA associated leukemic transcription program. However, it remains unclear whether and how HOTTIP lncRNA is involved in remodeling leukemic genome to facilitate AML leukemogenesis. Here, we showed that HOTTIP regulates a fraction of CTCF binding sites (CBSs) in the AML genome by directly interacting with CTCF and its binding motifs. We carried out CTCF ChIP-seq and HOTTIP ChIRP (chromatin isolation by RNA purification)-seq comparing WT and HOTTIP knockout (KO) MOLM13 cells. KO of HOTTIP in MLL-rearranged MOLM13 AML cells specifically impaired CTCF binding sites that were co-occupied by HOTTIP lncRNA, whereas loss of HOTTIP did not affect global CTCF binding. These target genes include posterior HOXA genes and Wnt target genes such as C-MYC, EVI1, AXIN, and TWIST1. Furthermore, we found that HOTTIP interacts with its putative target sites by formation of DNA: RNA hybridization structure triple helix and R-loop in vivo and in vitro. We then carried out DRIP (DNA-RNA immunoprecipitation)-seq and DRIPc(DNA-RNA immunoprecipitation followed by cDNA conversion)-Seq, which utilize a sequence independent but structure-specific S9.6 antibody for DRIP to capture global R-loops, by comparing WT and HOTTIP KO MOLM13 cells. The obtained DRIP-seq and DRIPc-seq data were then incorporated and integrated with the HOTTIP ChIRP-seq and CTCF ChIP-seq data to explore global collaboration between R-loop and HOTTIP associated CTCF binding sites. We found that HOTTIP interacts with CTCF binding motif that defines the TADs and the promoters of the HOTTIP target genes by formation of R-loop or triple helix structure. Loss of HOTTIP disrupted the R-loop formation at promoters and enhancers of the HOTTIP target genes to inhibit their expression. In MLL-rearranged AML genome, in addition to the HOXA locus, CTCF forms leukemic specific TADs that protect aberrant Wnt target genes. Depletion of HOTTIP lncRNA impaired CTCF defined TADs in the Wnt target gene loci and reduced Wnt target gene expression. In contrast, overexpression of Hottip lncRNA (Hottip-Tg) in the mice bone marrow hematopoietic compartment perturbs hematopoietic stem cell (HSC) self-renewal and differentiation leading to AML like disease by reinforcing CTCF defined TADs, enhancing chromatin accessibility within TADs, and upregulating gene transcription in the Wnt target loci. Finally, when we treated HOTTIP expressed primary patient AML cells carrying MLL-rearrangement and their derived PDX mouse model with a canonical Wnt inhibitor, ICG-001, ICG-001 inhibited AML LSC self-renewal in in vitro by LTC-IC assays and in vivo leukemogenesis in the PDX mouse models with an aberrant HOTTIP lncRNA expression, but not in HOTTIP negative/low non-MLL AML samples. Thus, HOTTIP lncRNA and CTCF cooperate to specifically reinforce CTCF defined WNT target locus TADs and drive Wnt target gene expression in the HOTTIP expressed AML. Disclosures No relevant conflicts of interest to declare.

A Phase Ib/II Study of the Histone Methyltransferase Inhibitor Pinometostat in Combination with Azacitidine in Patients with 11q23-Rearranged Acute Myeloid Leukemia

A Phase Ib/II Study of the Histone Methyltransferase Inhibitor Pinometostat in Combination with Azacitidine in Patients with 11q23-Rearranged Acute Myeloid Leukemia

Spliceosomal Disruption of the Non-Canonical SWI/SNF Chromatin Remodeling Complex in SF3B1 Mutant Leukemias

Mutations in the RNA splicing factor SF3B1 are common in MDS and other myeloid malignancies. SF3B1 mutations promote expression of mRNAs that use an aberrant, intron proximal 3' splice site (ss). Despite the consistency of this finding, linking aberrant splicing changes to disease pathogenesis has been a challenge. Here we identify aberrant splicing and downregulated expression of BRD9, a member of the recently described ATP-dependent non-canonical BAF (ncBAF) chromatin remodeling complex, across SF3B1 mutant leukemias. In so doing, we identify a novel role for altered ncBAF function in hematopoiesis and MDS. To systematically identify functionally important aberrant splicing events created by mutant SF3B1, we integrated differential splicing events in SF3B1 mutant versus wild-type MDS with a positive enrichment CRISPR screen mimicking splicing changes induced by mutant SF3B1 that promote NMD (non-sense mediated mRNA decay). We tested whether loss of any gene functionally inactivated by SF3B1 mutations promoted transformation of Ba/F3 and 32D cells. This identified a specific NMD-inducing aberrant splicing event in BRD9 which promoted cytokine independence (Fig. A) and exhibited striking aberrant splicing across CLL and MDS and across all mutational hotspots in SF3B1 (Fig. B). SF3B1 mutations cause exonization of a normally intronic sequence in BRD9, resulting in inclusion of a poison exon that interrupts BRD9's reading frame (Fig. C) and reduced BRD9 mRNA and protein expression through NMD (Fig. D). We confirmed that mutant SF3B1 suppressed full-length BRD9 levels without generating truncated BRD9 protein. Loss of BRD9 impaired ncBAF complex formation as indicated by abolished interaction between the ncBAF specific component GLTSCR1 and the ATPase subunit BRG1 upon chemical or spliceosomal BRD9 ablation (Fig. D). Given that prior work has linked mutant SF3B1 to use of aberrant 3' ss, we sought to understand the molecular basis for aberrant exon inclusion in BRD9 by mutant SF3B1. Lariat sequencing of SF3B1 mutant versus WT K562 cells and BRD9 minigene analyses identified use of a deep intronic branchpoint adenosine by mutant SF3B1 to promote BRD9 poison exon inclusion (Fig. E). The data above suggest a role for BRD9 downregulation in SF3B1 mutant leukemia. While prior work indicated that BRD9 is required in MLL-rearranged AML (Hohmman et al. Nature Chemical Biology 2016), the role of BRD9 in normal hematopoiesis or other subtypes of myeloid neoplasms has not been evaluated. Genetic downregulation of BRD9 in normal human hematopoietic progenitors from cord blood promoted myelopoiesis while impairing megakaryopoiesis. Interestingly and unexpectedly, BRD9 loss in CD34+ cells promoted terminal erythroid differentiation in vitro. To further evaluate BRD9's role in hematopoiesis in vivo, we also generated mice with inducible knockout of the bromodomain of BRD9 (required for BRD9 function) and generation of a frameshift transcript resulting in reduced Brd9 expression (Fig. F). Loss of Brd9 resulted in macrocytosis with bone marrow erythroid dysplasia in a dosage-dependent manner, along with impaired lymphopoiesis and myeloid skewing. Moreover, competitive transplantation of hematopoietic precursors from these mice revealed that ablation of Brd9 function impaired lymphoid reconstitution while promoting advantage of myeloid cells and hematopoietic precursors (Fig. G-I). In myeloid leukemia cells, introduction of SF3B1K700E or downregulation of BRD9 resulted in increased chromatin accessibility at promoters with a significant overlap in commonly upregulated genes. This finding suggests shared epigenetic effects of SF3B1K700E mutations and BRD9 loss (Fig. J). These data identify aberrant splicing of BRD9 across the spectrum of SF3B1 mutant cancers and identify a novel role for downregulation of ncBAF function in MDS pathogenesis. Consistent with human genetic data, genetic ablation of BRD9 function in mouse and human hematopoietic cells resulted in myeloid skewing and dyserythropoiesis. These data suggest that targeted correction of aberrant BRD9 splicing might serve as a novel therapeutic approach for SF3B1-mutant leukemias. Of note, treatment with drugs impairing the binding of mutant SF3B1 to RNA resulted in a dose-dependent rescue of aberrant BRD9 splicing in vitro (Fig. K) and in treatment of an SF3B1 mutant AML patient-derived xenograft in vivo. Figure Disclosures Kadoch: Foghorn Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Quantitative Analysis of MLL Fusion Transcripts By Droplet Digital PCR to Monitor Minimal Residual Disease in MLL-Rearranged Acute Myeloid Leukemia

Rearrangements of the mixed lineage leukemia gene (MLL, re-named KMT2A) result in aggressive leukemia. Current risk stratification of MLL-rearranged (MLL-r) leukemia is directed by the fusion partner gene and, increasingly, by minimal residual disease (MRD) assessment after induction therapy. The clinical significance of quantifying fusion transcript levels in leukemia patients is firmly established in chronic myeloid leukemia and acute promyelocytic leukemia but is less well studied in MLL-r patients. Real-time quantitative PCR (RQ-PCR) is the standardized assay for molecular MRD monitoring in patients with MLL-rearranged leukemia. However, this method is less precise when few leukemic cells are present, thus limiting its application for highly sensitive MRD monitoring. Droplet digital PCR (ddPCR) allows for absolute quantification of fusion transcripts when multiple copies of fusion transcripts are present per cell. Therefore, we aimed to evaluate whether determining MLL fusion transcript levels by ddPCR could improve the sensitivity of MRD monitoring in MLL-r leukemia.A total of 44 diagnostic and follow-up samples obtained from paediatric MLL-r leukemia patients (26 ALL, 18 AML) were subjected to targeted next-generation sequencing to obtain patient-specific fusion sequences. MLL fusion transcripts were quantified by ddPCR in a total of 17 samples obtained from 4 paediatric AML patients with MLL fusions involving MLLT3 (n = 3) and MLLT10 (n = 1). Fusion-specific probe assays were designed from each of the patient specific fusion sequences for MRD assessment by ddPCR. To determine the detection limit of this method in quantifying MLL fusion transcripts, two MLL-r AML cell lines (MV4-11 and THP-1), and one MLL-wt cell line (Kasumi-1) were used. MLL fusion transcript level of detection of ddPCR was determined by serially diluting MLL-r cDNA into MLL-wt cDNA (Kasumi-1). Using 20ng of MLL-r cDNA in 200ng diluent as the highest concentration, a 10-fold dilution series was performed to make concentrations ranging from 10−2 to 10−7. Each ddPCR reaction mixture contained 11ul of cDNA mix as template with 1X Supermix no dUTP (Bio-Rad), 500 nM of both F/R primers and 250 nM of 5'-FAM labelled probe (IDT). Droplets were generated using a QX200 Droplet Generator (Bio-Rad). A general thermal cycler protocol with annealing at 61°C for 1 minute was performed and positive fluorescence droplets were read using QX200 Droplet Reader (Bio-Rad). MRD of patient samples, derived from ddPCR, was then compared to MRD derived from DNA-based RQ-PCR, following the guidelines established by the EuroMRD group.Our ddPCR method showed high reliability and sensitivity, with the detection limit determined to be 10-5 for a cell line with low MLL fusion transcript expression (THP-1), and 10-6 for a cell line with high MLL fusion transcript expression (MV4-11). Comparison of results obtained by RQ- PCR and ddPCR in a total of 17 diagnostic and follow-up samples from 4 AML patients showed excellent/good concordance between methods for 13 samples with moderate MRD levels. The 4 samples with low levels of MRD (10-4 to 10-5) below the quantitative range as defined by EuroMRD for RQ-PCR were all detectable by ddPCR, highlighting that ddPCR could provide robust and highly sensitive MRD assays compared to the standardized RQ-PCR assays.In conclusion, ddPCR is a promising technique that can reproducibly and reliably quantify MLL-r transcripts for MRD monitoring of MLL-r leukemia. Highly sensitive and robust molecular MRD monitoring by ddPCR holds promise for improving response-based therapeutic stratification and prediction of molecular relapse before overt hematological relapse. DisclosuresNo relevant conflicts of interest to declare.

Molecular pathogenesis of disease progression in MLL-rearranged AML

Leukemic relapse is frequently accompanied by progressively aggressive clinical course. To understand the molecular mechanism of leukemic relapse, MLL/AF9-transformed mouse leukemia cells were serially transplanted in C57BL/6 mice (N = 96) by mimicking repeated recurrences, where mutations were monitored by exome sequencing (N = 42). The onset of leukemia was progressively promoted with advanced transplants, during which increasing numbers of somatic mutations were acquired (P < 0.005). Among these, mutations in Ptpn11 (p.G60R) and Braf (p.V637E) corresponded to those identified in human MLL-AML, while recurrent mutations affecting Msn (p.R295C) were observed only in mouse but not in human MLL-AML. Another mutated gene of interest was Gnb2 which was reported to be recurrently mutated in various hematological neoplasms. Gnb2 mutations (p.G77R) were significantly increased in clone size (P = 0.007) and associated with earlier leukemia onset (P = 0.011). GNB2 transcripts were significantly upregulated in human MLL-AML compared to MLL-negative AML (P < 0.05), which was supported by significantly increased Gnb2 transcript induced by MLL/AF9 overexpression (P < 0.001). In in vivo model, both mutation and overexpression of GNB2 caused leukemogenesis, and downregulation of GNB2 expression reduced proliferative potential and survival benefit, suggesting a driver role of GNB2. In conclusion, alterations of driver genes over time may play an important role in the progression of MLL-AML.

Abstract 4958: VTP50469 is a novel, orally available menin-MLL1 inhibitor effective against MLL-rearranged and NPM1-mutant leukemia

Abstract Inhibition of the menin(MEN) and MLL1(KMT2A) interaction reverses leukemic gene expression driven by MLL-fusion proteins and is a potential targeted therapeutic strategy in MLL-rearranged AML. Recent data show that inhibition of this interaction could also be efficacious in NPM1-mutant AML. We developed potent and selective menin-MLL1 inhibitors that are highly selective and effective in vitro and in vivo. Structure-based design yielded a potent small-molecule inhibitor of the menin-MLL1 binding interaction, VTP-49477 (Ki=12+5 pM). Human and mouse leukemia cell lines driven by MLL-fusion proteins or NPM1 mutations were selectively sensitive to VTP-49477 (IC50 of ~10 nM). Optimization of the inhibitor structure led to an orally available inhibitor, VTP-50469 (Ki=104+30 pM). Similar to VTP-49477, human cell lines and mouse primary cells carrying MLL-fusions or NPM1c-mutations were highly sensitive to treatment with VTP-50469 (IC50 ~20 nM). Mechanistically, treatment of NPM1-mutant OCI-AML3 cells and MLL-AF9 rearranged MOLM13 cells with VTP-50469 caused &amp;gt;20-fold reduction of menin in high-molecular-weight protein complexes as assessed by glycerol gradient sedimentation. Concordantly, VTP-50469 treatment resulted in &amp;gt;10-fold reduction in menin chromatin occupancy as assessed by ChIP-seq in MLL-AF4 rearranged RS4;11 and MOLM13 cells. Furthermore, treatment with either VTP50469 or VTP4777 reduced expression of common MLL-fusion target genes in RS4;11 and MOLM13 cells and also modulated gene expression in NPM1-mutant leukemia cells. Human PDX models were treated with either VTP-49477 or VTP-50469. Treatment of MLL-rearranged B-ALL and AML PDXs (n=3) with VTP-49477 for 28 days (50mg/kg, bid, IP) resulted in significant reduction of leukemia burden (median: 2-fold in BM, 3-fold in SP, and 6-fold in PB). For oral dosing, VTP-50469 was formulated in mouse chow (0.1%, total daily dose ~175mg/kg/day) and PDX models were treated with this chow for 28 days. Mice engrafted with MLL-rearranged B-ALL, MLL-rearranged AML and NPM1-mutant AML PDXs (n=5) showed dramatic reductions of human leukemia cells in peripheral blood, spleen and bone marrow as compared to control treated mice (median: 225-fold in BM, 14-fold in SP and 129-fold in PB). AML cells showed increased differentiation with elevated expression of CD11b, CD13, CD86 and loss of common MLL-fusion target gene expression. Treatment of MLL1wt and NPM1wt B-ALL and AML PDXs with VTP-50469 did not reduce leukemia burden, confirming VTP-50469 menin-MLL1 target selectivity. Treatment with VTP-50469 did not alter normal peripheral blood counts and no weight loss was observed during treatment. Therefore, at a highly effective dose, VTP-50469 does not have detectable toxicity. Based on these data, inhibition of the menin-MLL1 interaction with VTP-50469 may have beneficial activity in up to 40% of human AML and most infant leukemias. Citation Format: Andrei V. Krivtsov, Benjamin K. Eschle, Matthew Witkin, Jayant Y. Gadrey, Hannah J. Uckelmann, Sayuri Kitajima, Gerard M. McGeehan, Scott A. Armstrong. VTP50469 is a novel, orally available menin-MLL1 inhibitor effective against MLL-rearranged and NPM1-mutant leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4958.