Mutant AML Research Articles

Prognostic Impact of Clonality of Myelodysplasia-Related Gene Mutations in AML

Introduction: The 2022 International Consensus Classification (ICC) stresses the importance of molecular- and cytogenetic aberrations over previous medical history. Nine mutations are used to define a subgroup of AML with myelodysplasia related gene mutations (AML-MRGM). On a lower hierarchical level specific cytogenetic changes are used to define a subgroup of AML with myelodysplasia related cytogenetic abnormalities (AML-MRCA). Both groups together with TP53 mutations are categorized within the adverse risk group of the 2022 ELN classification. We evaluated the prognostic implications of clonality of MRGM mutations. Methods: 552 newly-diagnosed adult AML patients (pts.) with a median age of 55 years who received intensive induction chemotherapy followed by consolidation chemotherapy or allogeneic stem cell transplantation and with available genetic and follow-up data were included. Mutations present at diagnosis were identified by Illumina myeloid panel sequencing covering 48 AML-associated genes. Variant allele frequency (VAF) was adjusted for sex for mutations located on the X chromosome (BCOR, STAG2, ZRSR2). The prognostic VAF cutoff was identified by maximally selected rank statistics. We defined AML-MRGM as having at least one MRG mutation, AML-MRCA as having at least one MRCA without MRGM, TP53 mutated patients as having TP53 mutations regardless of the presence of MRGM or MRCA and for the remaining pts. de novo AML and secondary/therapy-related AML according to medical history of the patient. For multivariate analysis value imputation was used for missing values. Results:40 (7.2%) of 552 pts. were TP53 mutated,196 (35.5%) had at least one MRGM, and 47 pts. (8.5%) had MRCA. Among the clinically defined cases 215 pts. (38.9%) had genuine de novo AML and 54 (9.8%) had a previous history of a hematologic malignancy or cytotoxic treatment. The median follow-up was 5.8 years. Overall survival (OS) and relapse free survival (RFS) for de novo AML and secondary AML based on medical history were not significantly different and were therefore grouped together as clinically-defined AML (cdAML). AML-MRGM pts. had a significantly lower complete remission (CR) rate (OR=0.32, 95%CI 0.18-0.56, p<0.001) and shorter OS, but not RFS, compared to cdAML pts. (OS, HR 1.38, 95%CI 1.04-1.83, p=0.02; RFS, HR 1.12, 95%CI 0.84-1.48, p=0.44). AML-MRCA had similar OS and RFS compared to both cdAML and AML-MRGM pts., but a significantly better CR rate than AML-MRGM pts. (OR 5.68, 95%CI 1.64-38.65, p=0.01). Pts. with TP53 mutations had a worse OS and RFS than all other subgroups. A VAF of 45% was identified as a prognostic cutoff by maximally selected rank statistics. Patients with MRGM and VAF ≥ 45% were associated with higher age (p=0.04) and a lower platelet count at diagnosis (p=0.04). A VAF ≥ 45% was identified in 81 pts. (41.3%) and was associated with a significantly worse OS and RFS compared to the 115 pts. (58.7%) with a VAF below 45% (OS, HR 1.70, 95%CI 1.12-2.57, p=0.01; RFS, HR 1.65, 95%CI 1.05-2.57, p=0.03) (Figure 1). Patients with VAF ≥ 45% had an OS and RFS similar to AML-MRCA pts., but significantly shorter OS and RFS than cdAML pts. (OS, HR 1.89, 95%CI 1.32-2.69, p<0.001; RFS, HR 1.52, 95%CI 1.04-2.21, p=0.03). In multivariate analysis the presence of MRGM with a VAF ≥45% was an independent adverse prognostic factor for OS. Conclusion: In the present study, we confirm myelodysplasia-related gene mutations and TP53 mutations as negative prognostic markers in line with the hierarchical significance proposed by ELN. Our study suggests that the presence of MRGM is associated with worse OS especially if it is present at a VAF ≥ 45%, likely representing patients with at least one MRG mutation in the major AML clone.

Venetoclax and Azacitidine for Molecular Relapse during First Line Intensive Chemotherapy in Patients with NPM1 Mutated or Core Binding Factor (CBF) AML. a Study from the Dataml Registry

Introduction: Molecular follow-up (FU) for AML patients (pts) with NPM1 mutation, RUNX1::RUNX1T1 or CBFB::MYH11 fusion transcripts has been established over the last ten years. Real-time quantitative PCR (qPCR) is the gold standard for the evaluation of measurable residual disease (MRD) in pts undergoing intensive chemotherapy (IC). Failure to achieve a deep molecular response is a strong predictive factor of relapse and survival and may guide post remission therapy (Ivey A, NEJM 2016). Moreover, the ELN MRD working party recommended a schedule for molecular FU and recently established a definition of molecular relapse (Heuser M, Blood 2021). However, there is no therapeutic consensus for pts with molecular relapse before overt clinical relapse. Frontline allogeneic hematopoietic cell transplantation (alloHCT), salvage IC or even waiting for morphological relapse in order to screen molecular characteristics of the relapsing disease or propose a clinical trial, may be discussed with pts. Venetoclax with azacitidine (VEN/AZA) was recently approved in unfit AML pts and demonstrated a particular efficacy in NPM1-mutated AML. In this study, we thought to describe the safety and efficacy of VEN/AZA in pts with NPM1 mutated or CBF AML in molecular relapse after first line IC. Methods: Pts of the DATAML registry with the following criteria were included in this study : AML with NPM1 mutation, RUNX1::RUNX1T1 or CBFB::MYH11 transcripts, first line IC, morphological complete remission (CR), no alloHCT in first line, regular monitoring of MRD by qPCR in bone marrow (BM) and/or blood, molecular relapse defined according to ELN2021 guidelines on two successive samples, at least one cycle of VEN/AZA. Pts with molecular relapse followed by morphological relapse before VEN/AZA or those who relapsed after alloHCT were not included. Treatment: First line IC was cytarabine 200 mg/m² d1-7 with idarubicin 9mg/m²/d1-5 or daunorubicin 90mg/m²/d1-3 in pts 18-60y or cytarabine 100 mg/m² d1-7 with idarubicin 8mg/m²/d1-5 and lomustine 200 mg/m²/d1 in pts > 60y. Post remission therapy was intermediate or high dose cytarabine or mini-consolidations according to age and performance status. Midostaurin was added in FLT3-mutated pts. At time of molecular relapse, pts received off label venetoclax 400 mg/d (d1-14, d1-21 or d1-28 according to centers) with no ramp-up and azacitidine 75 mg/m²/d subcutaneously (d1-5, d8-9). Antifungal/antibiotic prophylaxis and G-CSF use were not systematically recommended. Results: Twenty-two pts treated with VEN/AZAfrom 21/09/2020 to30/03/2023 were included. Their characteristics at diagnosis were: female (n= 14), median age 55y (range 27-71), de novo AML (n=21), median WBC (68 giga/L, range 2.32-339.7), NPM1 mutation (n=20), CBFB::MYH11 (n=2), FLT3 mutation (n=13). All pts achieved first CR after one cycle of induction chemotherapy. Pts received a median of three consolidation cycles. All but three pts had negative MRD or low level MRD (LL-MRD) in blood and/or BM at the end of consolidation. The median time between CR1 and molecular relapse was 9.5 months (range, 1-50). The median time between molecular relapse and first VEN/AZA cycle was 51 days. During first cycle of VEN/AZA, 12, 5 and 5 pts received 14, 21 or 28 days venetoclax, respectively; 19 were treated as out-pts, 4 received posaconazole and GCSF was used in 11 pts. 12 and 3 pts had grade 3-4 neutropenia or febrile neutropenia (FN), respectively. Only 3 pts (14%) were hospitalized for FN. The median number of VEN/AZA cycles was 2 (range 1-12). After C1+/-C2, 10 (45%) and 11 pts (55%) had undetectable or LL MRD in blood, respectively (Fig1). 20 pts were bridged to alloHCT in morphological CR after a median of 2 cycles (range, 1-4). Only one pt had a molecular progression before transplantation after 2 cycles. Two pts died, one had a molecular relapse after 7 cycles and progress rapidly to frank relapse, the other one died of post-transplant complication while in CR with undetectable MRD. All other pts remained in CR at date of last news. With a median FU from D1C1 of 13.5 months, median OS wat not reached and 1y-OS was 86% (Fig2). Conclusion: In the setting of molecular relapse, VEN/AZA is safe, prevent overt relapse, and induce a high rate of molecular response before alloHCT. Molecular relapse becomes a major therapeutic challenge. Clinical trial endpoints should include the treatment of molecular relapse as an event for RFS and EFS estimation.

Outcome of Risk- and Response-Adapted Post-Remission Therapy in Patients with NPM1 AML Treated Intensively

Introduction: Molecular assessment of measurable residual disease (MRD) is a powerful prognostic tool to identify high risk of relapse. Specifically, large studies have shown that MRD negativity of NPM1 after two cycles of intensive chemotherapy (IC) predicts long term remission. The translation of MRD results to treatment decisions such as treatment augmentation (MRD erasing) and referral to an allogeneic stem cell transplant (allo-SCT) is still lacking. The purpose of this study is to report the outcome of AML patients with NPM1 mutation who received IC and achieved remission, in whom NPM1 MRD post cycle 2 of IC, combined with FLT3-ITD status, were used systematically to decide whether patients were to complete intensive consolidations or refer to alloSCT. Methods: This is a retrospective analysis from 3 large hospitals in Israel. Patients were treated with 7+3 for induction and a repeat induction based on day 14 marrow. Midostaurin was added for FLT3mut leukemia since 2018. Consolidation consisted of 3-4 HiDAC cycles at doses of 1-3 g/msqrX6. Gemtuzumab ozogamicin and maintenance with FLT3 inhibitors or CC486 could be added. NPM1 transcript levels (normalized to ABL copies) were collected at diagnosis, after 2 cycles of IC, at the end of IC cycles and during follow-up. Patients were transplanted if a donor was available and any of the following: NPM1 transcript level was not reduced by 3 logs by the end of second cycle chemotherapy, absolute level was >1%, poor risk AML (ELN 2017 guidelines) or high AR FLT3-ITD mutation was present. Patients were followed for relapse (type and timing) and whether a second remission was achieved, as well as for EFS and overall survival. Results: A database search yielded 90 patients with NPM1 AML who were treated according the abovementioned protocol between 5/2015 and 11/2022 and achieved remission, with a median follow-up of 872 days (range 103-2751). Forty-seven patients were to receive consolidation cycles without transplantation in CR1, based on the aforementioned criteria. All but one patient (death from sepsis) completed treatment cycles. During follow-up, 21 (45%) patients relapsed at a median of 343 days. Two patients died while in CR1 during follow-up (extensive burns; sudden death at home). In a subgroup analysis of patients <60y (N=28), there were 11 events (9 relapses and 2 deaths), whereas in patients >60y (N=19) there were 13 events (12 relapses and 1 death during follow-up). Of note, 4 patients >60y were treated with CC486, of whom only 1 relapsed. Of the 18 patients who relapsed and received further treatment (3 had palliative care only), only 1 patient did not enter CR2 (died during salvage). The other 17 patients entered CR2 after salvage regimen (N=7, 4 were qPCR MRDneg, 5 were subsequently transplanted in CR2) or after upfront alloSCT without any salvage regimen (N=10, 4 were qPCR MRDneg). Forty-three patients were planned for alloSCT at CR1 (27 - inadequate molecular response, 16 - ELN risk group). Only one of these patients failed to undergo alloSCT in CR1 as she relapsed. The other 42 patients underwent alloSCT at CR1. In this group of patients, the median progression-free survival was 5 years, with no difference in patients above or below the age of 60. One half of the events (9/18) were relapses and the other half were deaths in remission, almost all were GVHD associated (figure1). Discussion: NPM1 mut AML without FLT3 is still considered by the ELN to have favorable outcome and most patients are not being transplanted at CR1. Yet, many relapse. This study evaluated the outcome of AML patients according to molecular risk stratification and alloSCT consideration by the end of cycle 2 of IC. In line with previous publications, younger patients with standard-risk AML and with adequate molecular response had a favorable outcome, with >60% of them having a disease-free long-term survival without transplantation. Moreover, even in a case of relapse, patients can achieve CR2 with MRDneg and attain long-term remission (90% OS in long-term follow-up). Older patients have a less favorable outcome even when they achieve adequate molecular response with two thirds of them experiencing relapse. Hence, the potential long-term outcome without transplantation needs to be balanced by the high risk of relapse. Nevertheless, even these patients can be successfully treated with either a salvage regimen or immediate transplantation, and >40% of them can be long-term survivors.

BOD1L Facilitates Chromatin Binding of SETD1A and Promotes Leukemia Cell Growth

The H3K4 methyltransferase SETD1A plays an essential role in leukemia through a non-catalytic activity which regulates transcriptional pause release of RNA polymerase II. SETD1A localizes to the transcriptional start site of numerous actively transcribed genes, but primarily regulates the expression of a restricted set of genes within the DNA damage response as well as heme biosynthesis pathway. However, the roles of upstream regulators or essential components of SETD1A chromatin binding remain unclear. Using the DepMap database and a CRISPR-tiling screen method, we found that BOD1L is the most correlated co-dependency with SETD1A in human cancer cell lines and that the Shg1 domain of BOD1L plays an indispensable role in human MOLM-13 leukemia cell line. BOD1L knockout increases apoptosis and reduces cell cycle. The strong BOD1L dependency in AML cells was also confirmed using a mouse MLL-r leukemia model in vitro and in vivo. SETD1A catalytic domain mutant AML cells still responded to the BOD1L sgRNAs that suggests the role of BOD1L is independent from the SETD1A catalytic function. Importantly, BOD1L knockout mimics the transcriptional profiles of SETD1A knockout cells and reduces the expression of genes in DNA damage response and heme biosynthesis. BOD1L loss immediately reduces SETD1A distribution on chromatin and increased the NELFE and RNA polymerase II signals at transcriptional start sites. These results show that BOD1L is an essential component of the non-catalytic SETD1A function in transcriptional pause release. We also performed an immunoprecipitation assay using tagged constructs to examine the interaction between BOD1L and SETD1A. Our data indicate that SETD1A associates with BOD1L through the FLOS domain, especially in the C-terminal region. In addition, we found that the loop structure in the Shg1 domain of BOD1L is associated with the nonenzymatic domain of SETD1A. A split luciferase complementation assay reveals that the BOD1L Shg1 domain and SETD1A FLOS domain interact. To demonstrate the role of BOD1L chromatin interaction, we enforced localization of the Shg1 domain fragment at a silent gene locus and it successfully induced ectopic SETD1A recruitment to chromatin and formation of the COMPASS complex. However, we observed neither induced H3K4me3 modification nor RNA polymerase II recruitment. Taken together, while the BOD1L-SETD1A complex is not sufficient to act as a pioneer complex for active transcription, our study identified that BOD1L facilitates chromatin binding of SETD1A through the Shg1-FLOS domain interaction and then regulates transcriptional pause release, and that this interaction is a potential therapeutic opportunity for SETD1A-targeting therapy.

Combination of Decitabine and Etoposide Is Highly Effective in Treating p53-Mutated MDS/AML Via Activating Notch Signaling

The elderly MDS/AML patients, especially those with TP53 mutations, remain very difficult to treat and resistant to chemotherapy. A total of 105 elderly (age above 60 years) high risk MDS (n = 84, IPSS-R score > 4.5) or AML-MRC (n = 21) patients' records were collected from three medical centers across China and retrospectively analyzed. Decitabine and low dose etoposide was found to be superior to standard decitabine alone in MDS/AML patients with higher ORR (70.0% vs 52.7%) after two cycles of treatment, the longer median durations of ORR (24.0 months vs 12 months) and CR (38.0 months vs 24.0 months), and longer event-free survival (13 months vs 9 months, p = 0.023), without significant improvement in overall survival (20 months vs 15 months, p = 0.059). Other than the TP53 mutation, no other gene mutation (including NRAS, DNMT3A, TET2, EZH2, ASXL1, RUNX1) in the D+E group could reliably predict OS and EFS when compared to wild type phenotype. Patients with TP53 mutations showed higher response rate after two cycles of D+E therapy and had prolonged overall survival (Mutation vs WT: 31 months vs 9 months, p=0.012) and event-free survival (Mutation vs WT: 24 months vs 5 months, p = 0.022) compared with p53 wild type patients in D+E group from multivariate survival analyses. P53 mutated patients receiving D+E treatment also had significantly improved OS (31 months vs 12 months, p = 0.00) and EFS (24 months vs 8 months, p = 0.00) compared to patients receiving decitabine treatment. Moreover, we found decitabine and etoposide greatly reduced TP53 mutant AML tumor burden in the NSG mice xenografts (tumor burden: 92% reduction in p53-null xenograft mice, 65% reduction in p53-wild-type xenograft mice on day 20 after leukemia cells administration, p < 0.0001). It was found that TP53 mutant cells and p53 gene-editing MOLM13 cells showed terminal neutrophil differentiation in the combo of decitabine and etoposide, demonstrating the expected changes in cell-surface CD11b expression and neutrophil functional assays such as phagocytosis. Combo of decitabine and etoposide showed much higher percentage of differentiation marker expression compared to single drug decitabine or etoposide treatment. However, neither the p53 gene-edited THP-1 cells nor the TP53 wild type cell lines displayed markers of terminal differentiation, such as elevated CD11b expression or phagocytosis. Furthermore, the percentage of CD11b-positive cells increased significantly in p53-mutated primary cells while insignificantly in TP53 wild type cells. Decitabine and etoposide induced significant differentiation thus killing p53 mutated myeloid tumor cells and primary patients' cells in vitro. We also uncovered the underlying mechanism that why D+E could induce significant differentiation in TP53 Mutant while not in TP53 wild type myeloid tumors. Notch1 signaling pathway was activated after D+E treatment in TP53 Mutant myeloid tumors, which was consistently correlated with drug-induced myeloid differentiation. Notch1 knockout or Notch inhibitor incubation abrogated drug induced differentiation in TP53 Mutant myeloid tumors. The activation of the Notch1 signaling pathway has been observed to induce disease regression and decrease leukemia stem cells in AML mice as well as significant apoptosis in the AML patient sample. We also found D+E could upregulate transcription factor transcripts such as ETS1, ETS2, which could bind specifically to PSEN1 (PS1) promoter and activate its transcription directly. PS1 represent catalytic subunit of γ-secretase complexes and is responsible for Notch recognition. While in TP53 wild type AML cells, p53 might interact with p300. p300 acts as a coactivator of Ets1 and Ets2, competition for binding to p300 with p53 may confront p300 recruitment and repress ETS transcription activation directly, which might exert its effect on PS1 repression, thus inhibiting γ-secretase complex activity. In short, integrated p53 could repress PSEN1 transcription thus interfering gamma secretase complex activity, Notch recognition and cleavage as well as abrogating drug induced Notch activation and myeloid differentiation, which could be achieved in p53 mutated state. Combination of decitabine and etoposide regimen is also relatively safe and tolerable in elderly patients, which may open up a new path of therapy in the p53 mutated AML/MDS.

Development of a Fully Immunocompetent Adoptive Transfer In Vivo Model of Npm1 cA;Idh2 R140Q;Flt3 ITD AML

AML is a highly heterogeneous disease, both genetically and phenotypically. This includes both intra- and inter-patient heterogeneity that significantly impacts prognosis and treatment responses. Mouse models are a powerful tool to further our understanding of fundamental disease biology and to advance novel therapies. While human cell line (CDX) and primary patient (PDX) xenografts are reproducible in vivo models, these models are by necessity immunodeficient. Transgenic mouse models provide an opportunity to study disease development driven by human relevant mutations within a fully immunocompetent system. However, drug development studies often require large numbers of mice and predictable time of disease development that is infeasible using primary transgenic animals. We therefore sought to develop a transgenic mouse model representative of human disease that was able to consistently be adoptively transferred into fully immunocompetent recipients. Our laboratory has interest in multiple targeted therapeutics and chose to focus on a model that may have sensitivity to multiple translationally relevant targeted drugs. We crossed the previously established Npm1 cA (Vassiliou et al 2011), Flt3 ITD (Lee et al 2007) and Idh2 R140Q (Shih et al 2017) Cre-inducible alleles to generate a triple mutant AML model ( Npm1 cA;Idh2 R140Q;Flt3 ITD). This model develops an aggressive AML with a median survival of 44 days. Complete blood counts at endpoint demonstrate significantly elevated white cells as well as profound anemia and thrombocytopenia. Npm1 cA;Idh2 R140Q;Flt3 ITD mice also have splenomegaly and infiltration of immature myeloid cells into both lymphoid and non-lymphoid tissues. Flow cytometry analysis of the blood, spleen and bone marrow shows a significant expansion of CD11b+cKit+ cells characteristic of an AML blast phenotype. Given the aggressive nature of this model, we sought to determine whether Npm1 cA;Idh2 R140Q;Flt3 ITD AML cells could be adoptively transferred into either immunodeficient or fully immunocompetent recipients without prior preconditioning. NOD- Prkdc em26Cd52Il2rg em26Cd22/NjuCrl (NCG) and B6.SJL-Ptprca Pepcb/BoyJ (PepBoy) recipients were injected with 1x10 6 bulk splenocytes via the tail vein. Recipient mice quickly develop a lethal AML that faithfully recapitulates the disease observed in the primary transgenic donors. Circulating leukemia cells are detectable from approximately 2 weeks post adoptive transfer (post-AT) and median survival of PepBoy recipients ranged from 17 to 60 days depending on donor (Figure 1). Comprehensive immune profiling using a 36-specificity spectral flow panel showed that in PepBoy recipients, the donor derived cells accurately recapitulate the diversity of the AML immunophenotype of the primary transgenic donor. Notably, cells from male donors do not establish detectable disease in immunocompetent female recipients. This is associated with an increased number of T cells in the female recipients of male cells suggesting immune mediated control of AML development. To assess the response to targeted therapies in a fully immune competent model, we adoptively transferred of our Npm1 cA;Idh2 R140Q;Flt3 ITD to into PepBoy recipients. Mice were randomized to treatment groups based on peripheral blood disease burden at 14 days post AT (0.2-1% of CD45+ cells). Mice received daily oral gavage of either vehicle, 100 mg/kg venetoclax (BCL2 inhibitor), 100 mg/kg enasidenib (IDH2 inhibitor) or 30 mg/kg gilteritinib (FLT3 inhibitor). All three drugs resulted in a significant reduction in circulating tumor burden after 14 days of treatment. Enasidenib did not prolong survival compared to vehicle whereas venetoclax treatment resulted in a significant survival advantage (median 47 days vs 40 in the vehicle). This model was most sensitive to gilteritinib with a median survival of 68 days. (Figure 2). Together these findings demonstrated that the Npm1cA;Idh2 R140Q;Flt3 ITD model is an aggressive model of AML. Adoptive transfer of splenocytes into either immunodeficient NCG or fully immunocompetent PepBoy recipients faithfully recapitulates the disease of primary mice in a reproducible manner. This model therefore provides an in vivo model conducive to drug development studies and provides the opportunity to understand the impact of both AML development and therapeutics on the normal immune system.

Evaluation of Synergistic Anti-Leukemic Efficacy of PHI-101 in Preclinical Model of FLT3-ITD Acute Myeloid Leukemia

Background: AML is a heterogeneous disease with an overall cure rate of 40-50%. Some types of AML have even poorer prognosis with FLT3 mutant AML patients having only a 20-25% cure rate with chemotherapy alone. In order to improve the outcome for this high-risk AML, a number of tyrosine kinase inhibitors (TKI) have been developed and to date. Given the difficulty of identifying appropriate targets and treatment options for AML, combining drug options for AML treatment may produce clinical benefits. PHI-101 is a selective next-generation type I FLT3 inhibitor that shows potent anti-leukemic activity against AML samples harboring FLT3-ITD and/or TKD mutations. In a clinical trial, PHI-101 demonstrates significant clinical responses and tolerability in refractory or relapsed AML patients as monotherapy. To investigate the optimal way to combine PHI-101 with other therapies, a number of other drugs utilized in treating AML were tested both, in vitro and in vivo to determine synergistic or additive effects that might improve efficacy for relapsed/refractory/elderly FLT3 mutant AML patients. Study Design and Methods: Combination effects of simultaneous versus sequential treatment of PHI-101 with other agents, including daunorubicin, cytarabine, venetoclax (Ven), and azacytidine (Aza) were tested using human leukemia cell lines harboring FLT3-ITD mutations (MV4-11, Molm13, Molm14) or FLT3-wild type as controls. The cell growth inhibition was assessed by CCK-8, MTT and/or CellTiter-Glo assays as well as annexin V-binding by flow cytometry and the Chou-Talalay method was used to calculate the combination index (CI. using Compusyn software. The potential mechanisms of effect on combination therapy were explored by Western blotting of a variety of signal transduction pathways including FLT3 phosphorylation and downstream pathways BCL-2 family expression, and DNA damage. The MV4-11 xenograft mouse model was used to assess in vivo efficacy of PHI-101 combination therapy. Results: Among the substances tested for combination therapy with PHI-101 in FLT3-ITD cell lines MV4-11 and Molm14 cell lines, Aza and Ven treatment both showed synergy with both simultaneous and sequential treatment. Ven was highly synergistic when used simultaneously with PHI-101 in MV4-11 cells. Cell growth inhibition effect (GI 50) of PHI-101, Aza, and Ven used alone was measured first to calculate the CI in combination with increasing concentrations of each drug together of treatment for 72hrs. The GI 50 of PHI-101 in FLT3-mutant cell lines (MV4-11, Molm13, Molm14) were 7.0, 3.8, and 3.4 nM, respectively. The GI 50 of the gilteritinib was 5.7, 10.2, and 10.8 nM, respectively. On the other hand, the GI 50 of Aza were 198.9, 140.4, and 85.4 nM, that of Ven were 38.2, 8.4, and 56.5 nM. PHI-101 was the most efficient antitumor agent compared to other drugs tested in this study. The CI of Aza and PHI-101 was 0.01216 (EC 50), 0.05981 (EC 75), 0.29468 (EC 90) in MV4-11 cells. The observed synergistic effects were confirmed in the other FLT3-ITD cell lines as well. PHI-101 and venetoclax combined therapy in MV4-11 showed synergism (CI<0.1), and the CI was 0.00002 (EC 50), 0.00104 (EC 75), 0.01243 (EC 90). By Western blotting, the combination of Aza or Ven with PHI-101 resulted in a significant induction of p-H2AX and PARP cleavage in the FLT3-ITD cell lines (MV4-11), but not in FLT3-WT cell lines (THP-1). To further validate the synergistic anti-leukemic activity of PHI-101 with these agents, MV4-11-derived mouse xenograft models were generated and treated at various doses. In the MV4-11-xenograft model, PHI-101 alone or combined with Ven or Aza significantly decreased tumor volume. In line with the cell viability data, Ven and Aza combined with PHI-101 induced significant cell-killing in vivo. Conclusion: Treatment of AML cells with the combination of Ven or Aza with PHI-101 in vitro induced rapid induction of apoptosis and inhibition of cell growth that showed significant synergy. In vivo data showed improved efficacy for the combination treatment with these agents. In vitro and in vivo data from the preclinical AML models provides a rationale to evaluate the activity of PHI-101 in combination for patients who are ineligible for more intensive chemotherapeutic induction therapy. Improved anti-leukemic efficacy by combined therapy with PHI-101 represent a promising therapeutic strategy for newly diagnosed or R/R FLT3-mutant AML patients

Altered RNA Export in SF3B1 Mutants Increases Sensitivity to Nuclear Export Inhibition

SF3B1 mutations are the most frequent spliceosomal alterations across cancers, yet no successful therapy exists to target this pathway. Previous findings from a phase 2 clinical trial of the XPO1 inhibitor selinexor in patients with high-risk myelodysplastic syndrome (MDS) relapsed or refractory to hypomethylating agents (HMA) revealed increased activity in patients with SF3B1 mutations. XPO1 (Exportin-1) is responsible for the nuclear export of over 200 proteins, but also plays a role in the transport of multiple RNA species, including small nuclear RNAs (snRNAs), ribosomal RNAs (rRNAs), and select messenger RNAs (mRNAs) out of the nucleus. Given the role of XPO1 in exporting snRNAs, which form the catalytic portion of the spliceosome, we hypothesized that XPO1 inhibition may preferentially affect SF3B1-mutant cells via altered splicing, and that SF3B1-mutant high-risk MDS patients would have a better response to rational targeted drug combinations with next generation XPO1 inhibitors. To evaluate the mechanism underlying the preferential sensitivity of SF3B1-mutant cells to XPO1 inhibition, we performed nuclear and cytoplasmic fractionation followed by RNA sequencing (subcellular RNA-seq) before and after XPO1 inhibition in SF3B1 wildtype and SF3B1 K666N mutant cells. Whole transcriptomic analysis of subcellular RNA-seq data revealed increased global retention of RNA transcripts in the nucleus after XPO1 inhibition in the SF3B1 mutant cell line ( Figure 1). Validation assays using qPCR confirmed nuclear retention of several key transcripts such as Salt Inducible Kinase 1 (SIK1) and Cancer/Testis Antigen Family 45 Member A2 (CT45A2) in SF3B1-mutants (both SF3B1 K666N and K700E). Similarly, we performed subcellular RNA-seq for small RNAs and found snRNAs to be increased in the nucleus after XPO1 inhibition in the SF3B1 mutant cells. We then performed total cellular RNA sequencing to understand the effect of XPO1 inhibition on global RNA expression and RNA splicing. Differential gene expression analysis revealed that XPO1 inhibition had a more pronounced effect on cell cycle in SF3B1 wildtype cells, but differentiation pathways were more significantly affected in SF3B1-mutant cells. Alternative splicing analysis showed increased 3' alternative splicing events in SF3B1 mutant after XPO1 inhibition. These results suggest that the preferential sensitivity of SF3B1 mutant cells to nuclear export inhibition arises through increased nuclear retention of spliceosomal snRNAs and select mRNAs that results in perturbation of differentiation pathways. Despite the promising efficacy of XPO1 inhibition in SF3B1 mutated MDS, toxicity limits its dose escalation. Combination therapies may allow for greater synergism with lower doses of XPO1 inhibitor. In order to identify novel drug combination targets, we employed whole genome CRISPR screens in two myeloid leukemia cell lines treated with eltanexor, a next generation XPO1 inhibitor with lower toxicity than selinexor ( Figure 2). These analyses identified several novel targets, which we validated by testing for synergy in combination with eltanexor in wildtype and SF3B1-mutant cell lines. Using the Bliss independence model to calculate synergy, we identifed two drugs that greatly synergized with eltanexor specifically in the SF3B1 mutant cell lines: venetoclax (a BCL2 inhibitor), and navitoclax (a BCL2/BCL-XL inhibitor). We further validated these combinations in vivo using competitive transplant assays in Sf3b1 K700E conditional knock-in mice. The combination of eltanexor with venetoclax showed a significant decrease in the Sf3b1-mutant burden in the peripheral blood and bone marrow progenitor compartments, suggesting a preferential sensitivity of the combination for SF3B1 mutation. In conclusion, by analyzing the effects of XPO1 inhibition on RNA export, our study provides insight on the mechanisms behind the increased efficacy of XPO1 inhibition in SF3B1-mutant MDS/AML. Our findings may also contribute to the development of potentially synergistic therapeutic combinations. In this regard, recent human data have shown that venetoclax can overcome the poor prognosis of spliceosomal mutant AML patients (Senapati et al, Blood 2023); therefore, combining eltanexor with venetoclax could represent a promising SF3B1-specific therapy.

Forward Genetic Screen Implicates Drivers of Leukemic Progression in a Novel Model of Trp53 R270H myelodysplastic Syndrome

Myelodysplastic syndrome (MDS) is characterized by bone marrow failure and a highly variable clinical course. The most catastrophic complication of MDS is transformation to secondary acute myeloid leukemia (sAML). Notably, mutations in TP53 confer the single highest risk of transformation to sAML and death. However, some patients with TP53 mutated MDS do not develop sAML, suggesting that additional genetic events cooperate with TP53 mutations to transform MDS to sAML. Understanding the mechanisms of transformation of MDS to sAML could provide targets for therapeutic intervention. To model the genetics of MDS, we crossed mice bearing Trp53 R270H ( Trp53 is the murine TP53 gene) and deletion of genes syntenic with human chromosome 5q (del(5q)). To discover how additional mutations contribute to disease progression, we utilized Sleeping Beauty (SB) transposon mutagenesis in Trp53 R270H/del(5q) mice. SB transposase mobilized SB mutagenic T2/Onc transposons which randomly insert within the genome. T2/Onc transposons are designed to induce gain or loss of function alterations depending on the site and orientation of insertion with respect to targeted genes. We used the Mx1-Cre transgene to activate SB transposase and T2/Onc transposition in hematopoietic progenitors. Trp53 R270H anddel(5q)(or cytogenetically normal, CN) mice were crossed to SB mice to generate donor mice of the following genotypes: Trp53 R270H/del(5q)/ SB, Trp53 R270H/CN /SB, Trp53 WT/del(5q)/ SB, Trp53 WT/CN/ SB mice, and mice without SB transposition, (no transposition, NT: Trp53 R270H/del(5q) /NT). Bone marrow cells were transplanted into recipients, and SB insertional mutagenesis was activated using pI-pC to activate Cre. Mice receiving Trp53 WT/CN/ SB bone marrow developed more frequent T-cell leukemia (n=3/10) than myeloid leukemia (n=1/10). In contrast, mice receiving Trp53 R270H/del(5q)/SB and Trp53 R270H/CN /SB bone marrow developed predominantly myeloid leukemia (n=14/28) more commonly than T-cell leukemia (1/28). Mixed phenotype leukemia was seen in 7/28 of these mice. Together, these data demonstrate a strong bias towards myeloid disease in SB-mutagenized Trp53 R270H bone marrow. To identify genes with SB insertions, we performed RNA sequencing to detect SB T2/Onc transposon-endogenous genefusion transcripts. Among Trp53 WT/CN/ SB leukemias, the most common recurrent SB fusions involved Notch1 and Ikzf1 as has previously reported for SB-associated T-cell leukemias . Among Trp53 R270H/del(5q)/SB and Trp53 R270H/CN /SB leukemias, the most common recurrent SB-fusions involved Erg, Eras and Il2rb with Erg fusions detected 85% of Trp53 R270H leukemias (n=17/20). SB inserted upstream of Erg promoter indicating that these fusions likely upregulate expression of Erg. Indeed, Erg levels are significantly higher in leukemias that express SB-Erg fusions relative to leukemias that do not (p<0.0023). ERG is not recurrently mutated in human AML, but the ERG gene locus is commonly amplified, especially TP53 mutant AML. ERG is known to support normal hematopoietic stem cell self-renewal. Notably, Erg-insertions were also detected in a model of MDS expressing stabilized cyclin E with SB-mediated progression to erythroleukemia (Loeb 2019). Using gene set enrichment analysis, we found that hematopoietic stem cell and leukemic stem cell signatures are enriched in Erg-SB fusion leukemias. In our analyses of two independent data sets (TCGA and BEAT AML), stem cell signatures are also among the most highly enriched pathways in human AMLs expressing high ERG levels. Furthermore, in a human AML single cell RNA sequencing dataset (van Galen 2019), we found that ERG expression is highest in AML cells with the most immature stem and progenitor-like features. Together, these findings implicate a role for ERG as a driver of progression of MDS to AML by enhancing aberrant self-renewal. In summary, we present a novel murine model of Trp53/del(5q) MDS. In this model, Erg upregulation is associated with progression to AML and upregulation of leukemia stem cell gene expression profiles. These data implicate ERG as a major contributor to progression of MDS to secondary AML in the setting of mutant p53. Understanding the mechanisms of disease progression and self-renewal in myeloid malignancies with p53 mutations is critical to define effective therapeutic strategies in these rapidly fatal, treatment resistant diseases.

I-DATA Study: Randomized, Sequential, Open-Label Study to Evaluate the Efficacy of IDH Targeted/Non- Targeted Versus Non-Targeted/IDH-Targeted Approaches in the Treatment of Newly Diagnosed IDH Mutated Adult AML Patients Not Candidates for Intensive Induction Therapy

Background: The discovery of IDH1 and IDH2 mutations in AML, and the accompanying functional implications of the resulting neomorphic activity of these mutated enzymes, has resulted in FDA approved targeted therapies. Similarly, the changes brought about in methylation due to IDH mutations in the AML genome have ushered in treatment regimens of venetoclax with hypomethylating agents. In this setting, where traditional chemotherapy regimens also are applied for treatment of primary diagnoses, the available outcomes data for all three therapeutic approaches should now provide clear metrics of predicted response. However, given a lack of direct comparison studies, no guidance exists as to which treatment choice will provide best response in IDH-mutated patients. Furthermore, emerging data about the importance of the biologic context on the response to different agents, including co-existing gene mutations and patient age, pose additional questions that need to be systematically addressed in order to provide patients with the best treatment. Study Design and Methods: This is a randomized, multi-site Phase 2 study (NCT05401097) comparing overall treatment failure rates at 24 months between two sequences of first-line and second-line therapies in patients ≥ 18 years with newly diagnosed IDH1/2 mutated AML who are not candidates for intensive induction therapy (Figure 1). Patients are randomized into one of the two arms in a 1:1 ratio with stratification based on the type of IDH mutation. Randomized patients are then assigned to receive treatment sequence A (IDHi+Aza followed by ven+aza) or treatment sequence B (ven+aza followed by IDHi+aza). Five patients have been randomized and enrolled on study to date. Patients randomized to first-line IDHi will receive Ivosidenib 500mg po orally daily on Days 1-28 ( IDH1) or Enasidenib 100mg po orally daily on Days 1-28 ( IDH2), each of 28-day cycle. Aza will be given at 75mg/m 2 daily on days 1-7 or days 1-5/8-9 of each 28-day cycle. Patients can receive a maximum of 5 cycles to attain CR/CRh/CRi. Patients who achieve CR/CRh/CRi will continue IDHi+aza until unacceptable toxicity, withdrawal of consent for further protocol treatment, relapse, or death (henceforth referred to as 'cessation'). If a patient does not meet these parameters, progresses during the first 5 cycles, does not attain CR/CRh/CRi by the end of 5 cycles, attains CR/CRh/CRi by the end of 5 cycles and subsequently relapses, or has unacceptable toxicity, then the patient will stop therapy with IDHi+aza. For patients on second line Ven+aza, they may receive a maximum of 3 cycle of Ven+aza to achieve CR/CRh/CRi. Patients who achieve CR/CRh/CRi will continue Ven+aza until 'cessation.' If a patient progresses during the first 3 cycles, does not attain CR/CRh/CRi by the end of 3 cycles, attains CR/CRh/CRi by the end of 3 cycles and subsequently relapses, or has unacceptable toxicity, they will then come off study treatment. Patients randomized to first-line Ven+aza will receive ven dosing per the FDA-label. Aza will be given at 75mg/m 2 daily on days 1-7 of each 28-day cycle. Patients can receive a maximum of 3 cycles to attain CR/CRh/CRi. Patients who achieve CR/CRh/CRi will continue Ven+aza therapy until 'cessation.' If a patient progresses during the first 3 cycles, does not attain CR/CRh/CRi by the end of 3 cycles, attains CR/CRh/CRi by the end of 3 cycles and subsequently relapses, or has unacceptable toxicity, then the patient will stop therapy with the Ven+aza. For patients on second line IDHi+aza, they may receive a maximum of 5 cycle of IDHi+aza to achieve CR/CRh/CRi. Patients who achieve CR/CRh/CRi will continue IDHi+aza until 'cessation.' If a patient progresses during the first 5 cycles, does not attain CR/CRh/CRi by the end of 5 cycles, attains CR/CRh/CRi by the end of 5 cycles and subsequently relapses, or has unacceptable toxicity, they will then come off study treatment. Longitudinally embedded, comprehensive correlative studies will provide integrated genomic profiling of the leukemic clone during different disease stages and assess for potential resistance mutations or clonal evolution that may be predictors of relapse in this difficult to treat patient population. In addition, ultra-sensitive measurable-residual disease testing (ctDNA) and single-cell based combined transcriptional and mutational profiling will complement the detailed assessment.

Venetoclax Has Potent Efficacy in NPM1 mutated AML with Acquired Resistance Associated with Either Perturbed Pro-Survival Signalling or NPM1 wild-Type Populations

Background We have previously reported outcomes for patients (pts) receiving venetoclax (VEN) combined with intensive chemotherapy in fit older pts ≥65 years with newly diagnosed AML (Chua et al, JCO 2020). In contrast to younger populations, NPM1(mut) does confer favorable prognosis in older AML populations. Our prior studies indicate that VEN-based therapies in older pts have promising efficacy in NPM1(mut) AML (DiNardo et al, Blood 2020). To characterise this further, we performed a posthoc analysis of the efficacy of single agent VEN and in combination with chemotherapy in NPM1(mut) AML in the CAVEAT study and explored mechanisms of treatment failure using a multi-omic approach. Methods NPM1 MRD was performed using RT-qPCR (sensitivity 10 -6). Integrated analysis from bulk sequencing (targeted 42-gene NGS, whole exome/genome sequencing (WES/WGS), RNAseq), single cell (sc) proteogenomic and transcriptomic assessments (scDNA plus surface protein, Tapestri MissioBio; scRNAseq, 10x Genomics) were performed. Flow cytometry included analysis of BCL-2, BCL-XL, MCL1 and BFL1 expression. Results Of 85 pts enrolled on the CAVEAT study, 20 had NPM1(mut). Median age of NPM1(mut) vs (wt) was 71 vs 70 years. At a median follow-up of 38.6 months, CR/CRi rates and median OS for NPM1(mut) vs (wt) was 85% vs 72% (p=0.37), and 43.9 vs 15.1 months (p=0.04), respectively. Pts received a 7-day VEN monotherapy pre-phase prior to CAVEAT induction with paired bone marrow (BM) assessments pre- and at day 8 to evaluate VEN sensitivity. NPM1(mut) pts had a greater median relative blast reduction after 7-day VEN at -66% (range -7 to -98%), compared to -37% (range +51 to -94%) for NPM1(wt) (p=0.03). 18/20 NPM1(mut) sub-group pts completed induction and 17 had serial NPM1 MRD assessments. The NPM1(mut) level fell a median of 4.55-log 10 following induction, with 6 (35%) pts achieving MRD negative (MRDneg) status. After all consolidation, 12/16 (75%) were MRDneg. 12/12 (100%) pts in long-term follow up remain MRDneg. We investigated the mechanisms of resistance in the 5/20 pts with NPM1(mut) whose disease was primary refractory (RD; n=1) or had acquired secondary resistance (Fig). Two patients (CAL-045, CAL-021) had shown BM blast reductions of ≥45% during VEN pre-phase. Subsequently, at the time of clinical treatment failure, both were NPM1(mut) MRD negative indicating eradication of the starting NPM1(mut) clone and early evolution of a new NPM1(wt) population as the mechanism of resistance. Detailed genomic analysis of CAL-045 showed that a TET2/CBL/NPM1(mut) clone was eradicated, with persistence of TET2 and TET2/CBL(mut) clones lacking NPM1(mut) at the time of refractory disease. scRNAseq analysis of these NPM1(wt) clones revealed a pro-inflammatory signature with elevation in BFL1, a pro-survival gene not targeted by VEN (Fig). In patient CAL-021, NPM1(mut) was also absent with the emergence of nonsense/frameshift BAX variants at relapse as previously described (Moujalled et al, Blood 2022). BAX deficiency is associated with resistance to BH3-mimetics. Two other cases (CAL-014, CAL-020) showed BM blast reductions of <45% during VEN pre-phase. In CAL-014, the expansion of an NPM1 subclone was associated with FLT3-ITD. WES revealed evolution of FLT3-ITD loss of heterozygosity at relapse. Flow cytometric analysis demonstrated disproportionate upregulation of intracellular MCL-1 (Fig). In case CAL-020, no obvious genetic determinants of resistance were identified at relapse, with persistence of a DNMT3A/ KIT/ NPM1(mut) clone (VAF~50%). Whole transcriptome analysis revealed skewed expression of a KIT(mut)allele (from 45% to 88%) at relapse, and protein assessment by flow cytometry revealed marked upregulation of BCL-XL (Fig). Conclusion VEN in combination with intensive chemotherapy for NPM1(mut) AML pts ≥65 years resulted in rapid and deep responses ( NPM1(mut) MRD-negativity) that are associated with prolonged remissions and OS. Mechanisms of treatment failure among pts with NPM1(mut) are diverse and may include kinase-linked upregulation of alternative BCL-2 family pro-survival members or disruption of downstream pro-apoptotic BAX.

Characterization of Cases with the Rare Cytogenetic Abnormality I(7)(p10) Reveals an Association with IDH2 Mutated Acute Myeloid Leukemia

Background: The chromosomal alteration i(7)(p10) is a rare abnormality in hematological malignancies. It leads to a loss of the long arm (7q) and duplication of the short arm (7p) of chromosome 7. However, the presence, frequency and clinical implications of i(7)(p10) in hematological neoplasms has not been analyzed systematically to date. Aim: (1) Analysis of the frequencyand occurrence of i(7)(p10) in hematological malignancies. (2) Characterization of additional cytogenetic alterations and co-occurring mutations. (3) Analysis of potential clinical implications. Patients and methods: All samples sent for diagnosis of suspected hematologic neoplasms between 08/2005 - 05/2023 were used for detection of i(7)(p10). Presence of i(7)(p10) was analyzed using chromosome banding analysis (CBA). In cases with available molecular genetic analyses the following genes were analyzed: ASXL1, BCOR, CEBPA, DNMT3A, EZH2, FLT3-ITD, IDH1, IDH2, KRAS, NPM1, NRAS, RUNX1, SF3B1, SRSF2, STAG2, TET2, TP53, U2AF1 and ZRSR2. Variants of unknown significance were excluded from statistical analysis. All reported p-values are two-sided and were considered significant at p<0.05. Results: In 34 cases an i(7)(p10) was detected. Regarding specific entities, i(7)(p10) was mainly found in AML (23/34, 68%) and rarely in other entities (mature B-cell neoplasms (B-NHL): 6/34, 18%; MDS: 4/34, 12%; ALL: 1/34, 3%). In 20/34 cases, i(7)(p10) comprised the sole cytogenetic abnormality, including 19 AML cases and 1 MDS case. In 10/34 cases, additional chromosomal alterations were detected, leading to a complex karyotype in 5 of these cases. Of note, all cases with a complex karyotype were B-NHL. Additionally, in 4 cases i(7)(p10) was present only in a subclone (1 AML, 1 B-NHL, 2 MDS cases) (Figure A). In 18/34 cases, molecular genetic data was available, revealing DNMT3A (15/18), IDH2 (14/18) and BCOR (7/18) as most frequently mutated genes. As these 18 cases comprised n=15 cases diagnosed as AML, further analysis focused on these patients only. 14/15 cases harbored a mutation in IDH2, the remaining case showed an IDH1 mutation. Further, in 14/15, 7/15 and 4/15 cases, mutations in DNMT3A, BCOR and EZH2 were detected, respectively. By contrast, mutations that are known to frequently co-occur with IDH2 mutations in AML ( SRSF2, ASXL1, NPM1, STAG2, RUNX1, NRAS) were absent or only very rarely detected (Figure B). 12/14 cases with IDH2 mutations showed the mutation site p.Arg172Lys, 2/14 cases p.Arg140Leu, which is known to be the most frequent IDH2 mutation site. This is corroborated by analysis of a control cohort of n=60 IDH2 mutated AML cases without i(7)(p10), in which only 8/60 cases with p.Arg172Lys were detected (86% vs. 13%, p<0.001). Further, comparison of variant allele frequency (VAF) of IDH2 with the VAF of co-mutations suggested the presence of IDH2 in the main clone of all cases, although IDH2 VAF of cases with i(7)(p10) was slightly but not significantly lower than the IDH2 VAF of AML cases without i(7)(p10) (38.3 vs. 45.9). Moreover, comparison of IDH2 mutated AML with and without i(7)(p10) revealed a predominance of females (13/14, 93% vs. 29/60, 48%, p<0.001) and of younger age (63 vs. 72 years, p=0.015) in cases with i(7)(p10). Analysis of overall survival (OS) did not reveal a significant difference between IDH2 mutated AML cases with and without i(7)(p10), although cases with i(7)(p10) showed a trend towards a better outcome (37 vs. 14 months). Conclusions: (1) The rare but recurrent cytogenetic aberration i(7)(p10) is mainly found in AML, where associations with mutations in IDH2, DNMT3A and BCOR were observed. By contrast, other AML-defining mutations ( NPM1, FLT3-ITD, CEPBA) were largely absent, further no defining fusions/rearrangements or a complex karyotype were detected. (2) The vast majority of patients were female and younger than patients with IDH2 mutation without i(7)(p10). (3) Almost all cases showed the rare hotspot p.Arg172Lys and i(7)(p10) as sole aberration, thus it can be speculated that these cases define a distinct subgroup. (4) OS seems to be slightly better than in IDH2 mutated AML without i(7)(p10), although this difference was not statistically significant. (5) Taking into account that the IDH2 mutation was always detected in the main clone and that no other targetable genes/aberrations were observed, these patients are potentially suitable for targeted therapy with IDH inhibitors.

Long-Term Real-World Experience of CPX-351 in Combined French-Italian Cohorts Identified High Rate of Negative Measurable Residual Disease (MRD) and Prolonged Overall Survival

Introduction CPX-351 is a liposomal formulation of cytarabine and daunorubicin packaged at a 5:1 molar ratio. This drug has been approved by the FDA and EMEA for patients with therapy-related acute myeloid leukemia (t-AML) or AML with myelodysplasia-related changes (MRC-AML) according to the WHO 2016 AML classification. Real world experience from several countries (France, Italy, Germany, UK and US) showed similar results of phase 3 clinical registration but with short follow-up. Lancet et al. published a long-term update of phase 3 clinical trial and confirmed long-term remission and improvement of overall survival (OS) ( Lancet Haematology 2021). Thus, the primary objective of this study was to analyze the efficacy of CPX-351 in a real-life setting with a longer follow-up, evaluating the impact of measurable residual disease (MRD) in responding patients. Methods We retrospectively collected data from patients treated with CPX-351 in thirty-six centers in France and Italy. Overall response rate (ORR) was defined by complete remission (CR) and CR with incomplete hematological recovery (CRi). Among the patients in CR or CRi, 62 (61%) had MRD evaluation assessed by next-generation sequencing (NGS) or flow cytometry. OS was calculated from the date of AML diagnosis to the date of death or last follow-up. All statistical analysis were performed using SPSS v.22 software (IBM SPSS Statistics). Results Between April 2018 and October 2019, 170 patients treated with CPX-351 were included in this study. The sex ratio male/female was 80/90 and the median age was 66 years (range 20-83). AML subtypes were MRC-AML in 117 (69%) and t-AML 48 (28%) patients. According to the European LeukemiaNet (ELN) 2017 classification, genetic risk was favorable, intermediate, and adverse in 8 (5%), 61 (36%), and 98 (58%) (missing for 3 patients), respectively. According to the ELN 2022 classification, genetic risk was favorable, intermediate, and adverse in 8 (5%), 16 (9%) and 143 (84%), respectively. Thirty percent and 19% of patients had complex and monosomal karyotypes, respectively. Assessed by NGS the most frequent mutated genes were: TP53 (n=35, 21%), RUNX1 (n=30, 18%), ASXL1 (n=22, 13%) and TET2 (n=18, 11%) among the XX were NGS was available (sinon ca fait des % qui ne tombe pas juste sur les 170 pts). According to a genetic ontogeny-based classifier (Lindsley et al., Blood 2015), 28 %, 39% and 33% had de novo/pan-AML, secondary type mutations AML, and TP53 mutated AML, respectively. The ORR was 102/170 (60%) after one (n=91) or two (n=11) inductions including 53% CR and 6% CRi. Among the 102 CR/CRi patients, 62 (61%) were evaluable for MRD after induction or after first consolidation. Forty (65%) patients had MRD below the threshold of 10 -3. ELN2017 and ELN2022 were identified as factors predicting CR/CRi rate ( P=0.032 and P=0.043, respectivelyà but the Lindsley classifier did not predict response ( P= 0.060). After 3-years of follow-up, in a univariate analysis, only MRD >10 -3 ( P=0.031); ELN 2017 classification ( P=0.027) and presence of TP53 mutation ( P=0.017) but not ELN 2022 or the Lindsley classifier; were associated with a significantly poorer median OS. In a multivariate analysis, only MRD >10 -3 was associated with a poorer OS (hazard ratio [HR]=2.6, 95% CI 1.2-5.5, P=0.013). Median OS was 40.9 months vs. 10.6 months for patients with MRD <10 -3 vs. ≥10 -3, respectively ( P=0.006). Sixty (35%) patients underwent an allogeneic hematopoietic stem cell transplant (HSCT) with an improved median OS compared to non-transplanted patients (not-reached vs. 9.1 months, P<0.0001). We also observed a trend towards a better median OS in transplanted patients who underwent an HSCT with MRD <10 -3 (not reached vs. 26.0 months, P=0.06). Conclusion After 3 years of follow-up, the improved OS with CPX-351 was confirmed in the real- life setting. Achievement of MRD negativity contributed to improvement of OS in the overall population and, maybe, in transplanted patients. These data provide the rationale for the ongoing ALFA-2101 phase III clinical trial evaluating CPX-351 vs. 3+7 (daunorubicin and cytarabine) in non-MRC-AML and non-t-AML using MRD as the primary endpoint.

The Benefit of Allogeneic Transplant in 1 st Complete Remission in NPM1 Mutated AML with or without FLT3 ITD Is Restricted to Those Testing MRD Positive after Induction - an Analysis of the UK NCRI AML17 and AML19 Studies

Background While there is accumulating evidence that measurable residual disease (MRD) negative patients with intermediate risk AML may not benefit from allogeneic transplantation in 1 st complete remission (CR1-allo), many consider the presence of a FLT3 Internal Tandem Duplication (ITD) an indication for this procedure. However, the studies supporting this practice were mainly performed before the development of sensitive molecular MRD techniques (Schlenk NEJM 2008).The UK NCRI AML17 and AML19 trials studied, then incorporated, MRD by RT-qPCR into the management of patients with NPM1 mutated AML. Methods NCRI AML17 (April 2009 to December 2014) and AML19 (November 2015 to November 2020) were sequential prospective randomised trials for younger adults (18 - 60 years) with newly diagnosed AML. In AML17, decisions regarding CR1-allo were made using a validated risk score incorporating baseline factors and response to induction. In AML19, for patients with NPM1 mutated AML ( NPM1mut) the protocol recommended CR1-allo only in patients testing MRD positive in the peripheral blood (PB) post cycle 2 (PC2), regardless of other baseline risk factors such as FLT3 ITD, based on findings from AML17 (Ivey et al, NEJM 2016). Both trials were performed prior to the availability of midostaurin and FLT3 ITD MRD assays. Here we compare the outcomes of patients with NPM1mut in these trials and assess the benefit of CR1-allo according to FLT3 ITD and molecular MRD status. Time-to-event variables are analysed using the Kaplan-Meier method, and the impact of alloSCT assessed using time-dependent Cox regression and visualised with Simon-Makuch plots. Molecular relapses were included as relapse events, and alloSCT performed after molecular relapse was not considered as CR1. Results 737 patients with NPM1mutAML who achieved CR/CRi and had a valid PC2 PB MRD result were included in the analysis. 60/348 (17%) patients from AML17 and 83/389 (21%) from AML19 tested PB PC2 MRD positive. Overall, CR1-allo was performed in 20% of patients in AML17 and 25% in AML19. Of the MRD positive patients CR1-allo was performed in 16/60 (27%) in AML17 compared to 50/83 (60%) in AML19 where these patients were recommended for transplant. CR1-allo was performed in 18% and 16% of MRD negative patients in AML17 and AML19 respectively. In AML17, as previously reported (Ivey et al, NEJM 2016), patients who were MRD+ had poor outcomes, with 3-year cumulative incidence of relapse (CIR) of 84% and overall survival (OS) of 25%. These outcomes were improved in AML19, with 3y CIR 50% and 3y OS 51%. Across both trials, CR1-allo was associated with a significant survival benefit in MRD+ patients (3y OS 61% vs 24%, HR 0.42, 95%CI 0.26-0.69, p<0.001, Figure A). Patients who were MRD negative had excellent outcomes in both trials (3y CIR 33% and 24% and 3y OS 75% and 83% for AML17 and AML19 respectively). AlloSCT in CR1 provided no survival advantage for MRD- patients (HR 0.83, 95%CI 0.51-1.34, p=0.4). There were a total of 286 patients with NPM1mutFLT3 ITD mut AML with a PC2 PB MRD result across both trials. 81/286 (28%) were PB PC2 MRD+ of whom 32/81 (40%) received CR1-allo (28% in AML17 and 49% in AML19). Outcomes in these high-risk patients were improved in AML19, with 3y CIR 91% vs 68% and 3y OS 22% and 31% for AML17 and AML19 respectively. Those who received CR1-allo had improved outcomes (3y OS 45% vs 18%, HR 0.52, 95% CI 0.29-0.93, p=0.03) consistent with the overall MRD+ population. In the 70% of NPM1mutFLT3 ITD mut AML patients who achieved PB PC2 MRD-, outcomes were encouraging in both AML17 (3y CIR 37%, 3y OS 75%) and AML19 (3y CIR 27%, 3y OS 80%). CR1-allo was performed in 20% (19% in AML17, 21% in AML19), with no survival benefit for transplant (HR 0.80, 95% CI 0.37-1.72, p=0.6, Figure B). There remained no benefit when restricted to those with high (>0.5) FLT3 ITD allelic ratio (HR 0.63, 95% CI 0.2 - 2.23, p = 0.50) and there was also no evidence of an interaction between allelic ratio and benefit from transplant for MRD negative patients (p=0.86). Conclusion Molecular MRD can be used to stratify decision making concerning post remission therapy in patients with NPM1mut AML. Patients who achieve MRD negativity in the peripheral blood by the end of course 2 are at low risk of relapse and we found no evidence of benefit from allogeneic transplant in CR1, including in the subset with FLT3 ITD. This finding contrasts with MRD+ patients where there was a significant survival benefit for CR1 transplant.

Gilteritinib or venetoclax in relapsed or refractory FLT3mut acute myeloid leukemia.

e19046 Background: FLT3 mutations in AML are associated with high relapse rates and shorter survival compared to FLT3 wild-type. After intensive chemotherapy (IC) failure, clinicians can choose between several salvage regimens; however, the optimal therapy sequence remains unclear. This study compares two salvage therapies: gilteritinib or venetoclax and a hypomethylating agent (HMA; decitabine or azacitidine). Methods: We retrospectively analyzed 129 patients with AML and mutated FLT3-ITD or FLT3-TKD. We identified those that failed first-line IC (CPX-351 or conventional 7+3 with or without midostaurin) and received salvage. We compared patients treated with gilteritinib or venetoclax + HMA as the next line of therapy from January 1, 2018 to January 1, 2022 at VCU Massey Cancer Center. To reduce confounding, no patient in the venetoclax group received gilteritinib in a subsequent line of therapy. We recorded baseline disease characteristics, genetic risk by ELN 2022, induction regimens, response, and survival. We used Fisher’s exact test for nominal data and Mann-Whitney for ordinal data. We analyzed survival by the Kaplan-Meier method and compared groups with the log-rank test. Results: We identified 129 patients with FLT3-ITD or FLT3-TKD mutated AML and analyzed them over 228 treatment phases. In the front-line setting, 89 (68.9%) patients received IC; 55 (61.8%) received salvage therapy for relapsed or refractory disease, and 16 received either gilteritnib (N=6) or venetoclax-based (N=10) salvage. The median age of the gilteritinib cohort was significantly younger ( p = 0.007), but there were no differences between groups with respect to ELN 2022 adverse risk ( p = 0.633) or between ECOG ( p = 0.700) or Charlson Comorbidity Index scores ( p = 0.104). The composite complete remission (CCR; CR + CRi) rate of the gilteritinib cohort was 20.0%, compared to 60.0% for the venetoclax cohort ( p = 0.282). No patient in the gilteritinib cohort achieved MRD negativity. Among responders in the venetoclax cohort, MRD negativity was 75.0% ( p = 0.400). The median overall survival in the gilteritinib cohort was 3.9 months — significantly shorter than 7.8 months for the venetoclax cohort ( p = 0.048). Conclusions: Venetoclax + HMA appears superior to gilteritinib for subsequent therapy after IC failure in FLT3mut AML, despite the gilteritinib cohort being significantly younger. The limitations of this study include its retrospective nature — prospective trials are needed to confirm these findings. [Table: see text]

Role of Gene Mutations in Acute Myeloid Leukemia: A Review Article

AbstractAcute myeloid leukemia (AML) is an immensely heterogeneous disease characterized by the clonal growth of promyelocytes or myeloblasts in bone marrow as well as in peripheral blood or tissue.Enhancement in the knowledge of the molecular biology of cancer and recognition of intermittent mutations in AML contribute to favorable circumstances to establish targeted therapies and enhance the clinical outcome. There is high interest in the development of therapies that target definitive abnormalities in AML while eradicating leukemia-initiating cells. In recent years, there has been a better knowledge of the molecular abnormalities that lead to the progression of AML, and the application of new methods in molecular biology techniques has increased that facilitating the advancement of investigational drugs.In this review, literature or information on various gene mutations for AML is discussed. English language articles were scrutinized in plentiful directories or databases like PubMed, Science Direct, Web of Sciences, Google Scholar, and Scopus. The important keywords used for searching databases is “Acute myeloid leukemia”, “Gene mutation in Acute myeloid leukemia”, “Genetic alteration in Acute myeloid leukemia,” and “Genetic abnormalities in Acute myeloid leukemia.”

Abstract A08: Targeting HSP90 epichaperome in TP53 mutant AML

Abstract The Bcl-2 inhibitor venetoclax (VEN)/hypomethylation agent (HMA) combination achieves high response rates, has improved outcomes for many patients with AML and is now considered standard of care for patients who are older or unfit to receive intensive chemotherapy. However, the median overall survival is only 14.7 months on this regimen and only 2.5 months post relapse. Molecular analysis demonstrates that mutations in TP53 and oncogenic kinases are key determinants of lower response rates and early relapse. Preclinical studies also show that increased kinase signaling in AML stem/progenitor cells in TP53 mutant AML. The heat shock protein 90 (HSP90) chaperone, a key regulator of proteostasis, is responsible for the correct folding of kinases and transcription factors. HSP90-associated-epichaperomes, formed in malignant cells, are complexes consisting of HSP90, co-chaperones, and associated proteins that support the maturation, activity, and stability of many cancer-associated kinases and transcription factors including mutated TP53. Hence, HSP90 epichaperome inhibition has the potential of targeting TP53 mutant AML. In contrast to other HSP90 ATP inhibitors, PU-H71 (zelavespib) is a competitive inhibitor specific for the ATP binding site of HSP90 epichaperomes. We here investigate the therapeutic potentials of targeting HSP90 epitherachorme with PU-H71 in TP53 mutated AML. Western blot analysis found increased HSP90 and several signaling proteins in TP53 knockout and mutant Molm13 cells generated by CRISPR/cas-9 or by exposing to idasanutlin, compared to the isogeneic wild-type controls. Using a fluorochrome-labelled PU-H71 and flow cytometry, we demonstrate the presence of HSP90 epichaperomes in AML cells and AML stem/progenitor cells with TP53 mutations, but not in normal bone marrow and bone marrow stem/progenitor cells. PU-H71 effectively kills AML cells and AML stem/progenitor cells with various TP53 mutations, and prolongs survival in TP53-mutant AML xenograft mice with minimal effects on normal CD34+ bone marrow cells and hematopoiesis. PU-H71 increased Bim expression and enhanced VEN activity in AML cells and AML stem/progenitor cells with TP53 mutations. Importantly, in a mixture of TP53 wild-type/R248W Molm13 cells (1000:1), nutlin3a selectively killed TP53 wild-type but enriched TP53 mutant Molm13 cells; VEN treatment favored the outgrowth of TP53-mutant cells, while PU-H71 effectively killed TP53 wild-type and mutant cells. Furthermore, PU-H71 exhibited anti-leukemia activity against both TP53 WT and mutant AML cells, which was further enhanced by VEN in vivo in a xenograft model of mixed TP53 WT and mutated Molm13 cells (10:1). Our data support that the HSP90 epichaperome is essential for the growth and survival of AML and AML stem/progenitor cells harboring mutant TP53. Inhibition of HSP90 by PU-H71 targets AML cells/stem/progenitor cells enhances VEN activity and prevents outgrowth of VEN-resistant TP53 mutant AML cells. This concept warrants clinical evaluations. Citation Format: Bing Carter, Po Yee Mak, Wenjing Tao, Lauren B Ostermann, Yuki Nishida, Steffen Boettcher, Michael Andreeff. Targeting HSP90 epichaperome in TP53 mutant AML [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A08.

Abstract A09: Targeting of FOXM1 recapitulates NPM1 mutations in chemo-sensitization of AML

Abstract NPM1 mutations are among the most common mutations in CN-AML (Cytogenetically Normal-AML) and are known to enhance chemosensitivity in AML patients. Across multiple co-operative group studies, in the absence of deleterious mutations, patients with NPM1 mutations show superior overall and relapse-free survival. The FOXM1 regulatory network is a major predictor of poor prognosis in human cancers of different origin, including AML. Early AML cell line and patient sample data from our lab demonstrated that nuclear NPM colocalizes with nuclear FOXM1 while mutant NPM sequesters FOXM1 in the cytoplasm. Applying RNAseq differential expression results from published FOXM1 knockout experiments on KG-1 cells and MLL-AF9-transformed mice to Beat AML RNAseq data, we developed a ~200-gene FOXM1 signature that was significantly associated with inferior CR/CRi rate (OR=0.44 [95% CI: 0.21–0.95], p=0.037) in wild type NPM1, wild type FLT3 de novo AML patients. Notably, this FOXM1 signature was not significantly associated with CR/CRi in mutated NPM1, wild type FLT3 patients (OR=1.78 [95% CI: 0.15–21.51], p=0.651). These data suggest that inactivation of FOXM1 may underlie the chemosensitivity conferred by the highly prevalent NPM1 mutation in favorable risk AML. Using our novel network-centric approach, we found a first-in-class small molecule (STL427944) that affects a pathway that limits FOXM1 expression and significantly disrupts the associated regulatory network. To directly address the role of FOXM1 in chemosensitivity of AML cells, we tested two FOXM1-specific inhibitory compounds: STL427944 and its derivative, STL001 (their structures will be disclosed). From a medicinal chemistry standpoint, STL427944 has two main metabolic liabilities, a hydrolytically labile furane-carboxylic acid phenolic ester and a hydrazone. We applied several paths of structural activity relationship (SAR) optimization to overcome these issues from removal of liabilities to bio-isosteric replacement to cyclizations. The ring replacement is likely to have significantly improved the overall stability observed in STL001, resulting in at least !0-fold more active compound with better ‘drug-like’ properties and thus enhanced potency. We showed that 500nM of STL001 partially suppressed expression of FOXM1 protein in AML, while 5mM of STL001 (versus 50mM of STL427944) fully suppressed FOXM1 and sensitized AML cells to venetoclax and cytarabine treatment. Ex vivo treatment of AML patient samples with STL427944 and STL001 resulted in inhibition of nuclear FOXM1 expression according to IHC and downregulation of canonical FOXM1 transcriptional targets as indicated by real-time PCR. This treatment potentiated the efficacy of venetoclax in inhibiting colony forming activity in AML patient samples. Our data suggest that targeting FOXM1 in AML patients with wild type NPM1 may recapitulate NPM1 mutations and will have a robust therapeutic effect leading to more favorable outcome for AML patients. Citation Format: Irum Khan, Sandjeev Raghuwanshi, Andy Kaempf, Jeff Tyner, Alex Domling, Carlos Camacho, Andrei Gartel. Targeting of FOXM1 recapitulates NPM1 mutations in chemo-sensitization of AML [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A09.

Abstract A31: C-MYC Targeting by Degradation: Novel Dual c-MYC/GSPT1 Degrader GT19715 Induces TP53-independent Cell Death in MYC-amplified Acute Myeloid Leukemia and Lymphomas

Abstract The oncoprotein c-MYC, a major regulator of the epigenome and transcriptome, is dysregulated in 70% of all human cancers. MYC is highly expressed in Burkitt lymphoma and TP53 mutant and venetoclax (ven) resistant AML (Sallman, Blood 2021, Nishida, ASH 2021). However, targeting c-Myc or the MYC pathway has not been successful and remains a major unmet clinical need. We developed the first cereblon E3 ligase modulators (CELMoDs) for c-MYC: GT19630 and GT19715 (salt form of GT19630). C-MYC was one of the top decreased proteins in chromatin-enriched proteomics, was pulled down by biotinylated GT19630 in in vitro affinity purification assay and was degraded with IC50 of 0.33 nM in MYC-amplified HL-60 cells. Proteasome inhibitor ixazomib completely blocked c-MYC reduction, suggesting a CRL4CRBN-dependent degradation. Blood cancer cell lines responded to GT19715 greater than other cancer cell lines such as lung, breast and brain tumors in a broad cell line panel, providing rationale to develop the degrader in hematologic malignancies. In agreement with other CELMoDs, proteomic analyses revealed degradation of translation termination factor GSPT1 (G1 to S phase transition proteins 1), an important factor in LSC survival Whereas a selective GSPT1 degrader CC-90009 reduced GSPT1 protein levels but not c-MYC, GT19715 reduced both c-MYC and GSPT1 and exerted a 20x higher cytoreduction than CC-90009 (IC50 of 1.8 nM vs 40.4 nM for GT19715 and CC-90009, respectively) in HL-60 cells. GT19630 degraded c-MYC and GSPT1 and inhibited tumor growth in a xenograft model with HL-60 cells. GT19715 eliminated circulating blasts and prolonged survival in the systemic Burkitt lymphoma model (Daudi). GT19715 significantly reduced human CD45+ AML blasts in peripheral blood, bone marrow (BM) and spleens compared to vehicle controls in vivo in a chemotherapy-resistant AML PDX model. MV4;11 venetoclax resistant (VR) cells demonstrated elevated protein levels of c-MYC and GSPT1 and GT19715 induced 4log10 cytoreduction in BM and prolonged survival of mice with MV4;11 VR cells. Baseline c-Myc protein levels associated with sensitivity to GT19715 in MOLM-13 cells with CRISPR engineered knockout/mutations of TP53 (R2 = 0.86, P = 0.02). GT19715 induced comparable cell death in primary AML samples with wild-type or mutant TP53 (95.4% and 91.7% cytoreduction, P = 0.48 for wild-type and mutant TP53 samples at 64 nM of GT19715, respectively). CD34+ AML cells were more susceptible to GT19715 than CD34- AML cells, suggesting a greater efficacy in AML stem/progenitor than in more mature AML cells. Notably, single cell mass cytometry revealed that CD34+ AML cells had higher c-MYC protein levels than CD34+ normal BM cells and GT19715 reduced c-Myc levels in CD34+ AML but not normal BM cells. GT19715 induced greater cytoreduction in CD34+ AML than in normal BM cells, suggesting a therapeutic window. Conclusions: The novel dual c-MYC/GSPT1 degrader GT19715 exerts TP53 independent preclinical anti-lymphoma and -leukemia efficacy, providing rationale for its clinical development. Citation Format: Yuki Nishida, Darah A Scruggs, Edward Ayoub, Lauren B Ostermann, Tallie Patsilevas, Vivian R Ruvolo, Po Yee Mak, Bing Z. Carter, Steffen Boettcher, Abhishek Maiti, Koji Sasaki, Qianxiang Zhou, Zhaohui Yang, Honghua Yan, Liandong Ma, Michael Andreeff. C-MYC Targeting by Degradation: Novel Dual c-MYC/GSPT1 Degrader GT19715 Induces TP53-independent Cell Death in MYC-amplified Acute Myeloid Leukemia and Lymphomas [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A31.

Impact of KRAS and NRAS mutations on outcomes in acute myeloid leukemia

The prognostic significance of RAS mutations in AML is poorly understood. In this ambispective cohort study of 239 newly-diagnosed AML patients at the University of Maryland, we assessed the median overall survival (mOS) and median event-free survival (mEFS) in RAS wild-type (WT) AML (n = 196), KRAS-mutated AML (n = 11), NRAS-mutated AML (n = 25), and KRAS/NRAS-mutated AML (n = 7). We used propensity score to adjust outcomes. NRAS-mutated AML had a similar response rate to first-line treatment and mOS compared to RAS-WT AML (57 vs. 54%, p = 0.8, 22.7 vs. 14.6 months, p = 0.7). The mOS of KRAS-mutated AML was shorter compared to RAS-WT AML (p = 0.049) and NRAS-mutated AML (p = 0.02). KRAS-mutated AML treated with anthracycline-based first-line regimens had a lower relative mortality compared to treatment with hypomethylating agents with venetoclax (HR <0.01, p = 0.04) and without venetoclax (HR <0.01, p = 0.04). This study demonstrates that KRAS but not NRAS mutations are associated with worse outcomes in AML. NOVELTY STATEMENT What is the new aspect of your work? The clinical significance of RAS mutations remains poorly defined and prior studies have yielded conflicting results. We used causal inferential methods, propensity score modeling, to determine the impact of KRAS and NRAS mutation on survival in newly diagnosed AML patients, independent of other risk factors. Moreover, we analyzed the outcomes of KRAS and NRAS-mutated AML patients receiving first-line therapy with hypomethylating agents and other non-anthracycline-based regimens. We provided a detailed description of RAS-mutated AML, including co-occurring mutations and cytogenetic abnormalities. What is the central finding of your work? KRAS mutations but not NRAS mutations in AML are directly linked to worse outcomes even after controlling for differences in AML type, co-occurring cytogenetic changes, treatment regimens, and comorbidities. KRAS-mutated AML has a higher relative mortality when treated with a hypomethylating agent-based first-line induction regimen compared to treatment with an anthracycline-based regimen. What is (or could be) the specific clinical relevance of your work? Our findings can help refine our genetic profiles of AML, improve prognostic models, and better stratify treatment regimens.