NPM1-mutated Acute Myeloid Leukemia Research Articles

Quantitative Impact of Mutated NPM1 MRD in Patients with AML Undergoing Allogeneic Stem Cell Transplantation

Introduction: In NPM1-mutated (NPM1mut) acute myeloid leukemia (AML) the detection of measurable residual disease (MRD) using quantitative PCR methods for NPM1mut allows for risk stratification and therapy adjustments during disease course. Even though it is well understood that a persistent NPM1mut burden at allogeneic stem cell transplantation (HSCT) is associated with relapse and shorter survival, the quantitative impact of NPM1mut at HSCT has not been investigated in detail. Methods: We analyzed 81 NPM1mut AML patients (pts) who received their first allogeneic peripheral blood HSCT at a median age of 61 (range 33-76) years in morphologic complete remission (CR) or CR with incomplete peripheral recovery (CRi). Conditioning regimens were non-myeloablative (70%), reduced-intensity (11%), or myeloablative (19%) according to EBMT guidelines. At diagnosis, cytogenetics and co-mutational status of FLT3, IDH1, IDH2, DNMT3A, and CEBPA were assessed. NPM1 MRD up to 28 days prior to HSCT in CR/CRi was measured by digital droplet PCR using a competitive probe approach, and results were normalized to ABL1 (limit of detection 0.001%NPM1mut/ABL1). For patients with a positive NPM1 MRD test result, receiver operator characteristics (ROC) curves were used to analyze distinct NPM1/ABL1 MRD threshold. Presence of chronic graft-versus-host disease (GvHD) was assessed according to Glucksberg criteria. Median follow up after HSCT was 4.4 years. Results: ROC curves identified 0.01% NPM1mut/ABL1 as the most informative cut-off to define MRDpos and MRDnegpts in both, bone marrow and blood, and irrespective of conditioning intensity. Using this cut-off 31 (38%) pts were NPM1mut MRDpos at HSCT and had a significantly higher cumulative incidence of relapse (CIR, P<.001, after 2 years 4% vs 66%) and shorter overall survival (OS, P<.001, after 2 years 77% vs 38%). Patients transplanted NPM1mut MRDpos had a 20.8-times higher risk of relapse than those transplanted MRDneg. While there were no differences regarding the gene mutation profile at diagnosis in pts with or without NPM1mut MRD at HSCT (Figure 1A), pts with NPM1mut MRD at HSCT had a significantly higher diagnostic WBC (49.6 vs 17.9; P=.03), LDH (12.9 vs 6.7; P=.02), and more CD34+ cells in bone marrow at diagnosis (1.4% vs 0.5%; P=.02). We observed a negative correlation of the NPM1mut /ABL1 MRD levels before HSCT and the time to relapse; the higher the NPM1mut MRD burden at HSCT the earlier the relapse occurred (R= -0.5, P=.03). This correlation was strongest in pts with a FLT3-ITD present at diagnosis (R= -0.82, P=.002). When utilizing two cut-offs for low (≥ 0.01% but < 1%) and high (≥ 1%) NPM1mut /ABL1 MRD levels there was a significantly shorter time to relapse in relapsing AML pts with high NPM1mut MRD levels (median time to relapse: 73 days) before HSCT compared to pts with low MRD levels (median time to relapse: 149 days). In addition, compared to patients with low NPM1mut MRD levels, pts with high NPM1mut MRD levels showed a trend for shorter OS (P=.10; OS after 2 years: 77% for NPM1mut MRDnegpatients vs 48% for low NPM1mut MRDpos patients vs 21% for high NPM1mut MRDpos patients; Figure 1B), indicating that pts transplanted with higher NPM1mut MRD may be harder to salvage. Presence of a FLT3-ITD and age at HSCT did not alter the negative impact on outcomes of low or high NPM1mut MRD at HSCT. We also evaluated the prognostic impact of the presence of chronic GvHD as a surrogate marker for graft-versus-leukemia effects with respect to the NPM1mut MRD status at HSCT. In a landmark analysis for pts surviving longer than 100 days, NPM1 MRDneg (< 0.01% NPM1mut/ABL1) pts had the lowest CIR (4% after 2 years), followed by NPM1mut MRDpos pts developing a chronic GvHD (25% after 2 years) and by NPM1 MRDpos pts who did not develop a chronic GvHD and had a very high CIR (92% after 2 years, P<.001). Conclusion: AML patients with > 0.01% NPM1mut/ABL1 MRD before allogeneic HSCT had a significant higher risk of relapse and shorter OS. High WBC, LDH, and CD34+ cells in bone marrow at diagnosis associated with persisting NPM1mut MRD at HSCT. The higher the NPM1mut MRD burden at HSCT, the earlier the relapse, and the lower the chances of successful salvage therapy after relapse. Development of a chronic GvHD after HSCT was associated with a reduced risk of relapse in NPM1mut MRDpos pts, indicating that bolstering immunologic effects post-HSCT for NPM1mut MRDpos AML pts may be a promising option to improve outcomes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Menin Inhibitor DS-1594b Drives Differentiation and Induces Synergistic Lethality in Combination with Venetoclax in AML Cells with MLL-Rearranged and NPM1 Mutation

Introduction: Mixed-lineage-leukemia (MLL) is one of the most frequently translocated genes in hematologic malignancies, which produces aggressive leukemias that are refractory to conventional treatment resulting in a 5-year survival rate of only 35%. MLL translocations lead to fusion of the N-terminal fragment of MLL with one of over sixty protein partners, producing MLL fusion proteins that enhance proliferation, block hematopoietic differentiation, and drive leukemia development. Acute myeloid Leukemia (AML) is a heterogeneous and rapidly progressing blood cancer with a poor prognosis, especially in elderly patients where it is more common. Mutations in the nucleophosmin (NPM1) gene are one of the most common causes of AML. Interestingly, the protein-protein interaction between Menin, MLL and MLL fusion proteins plays a critical role in the pathogenesis of acute myeloid leukemias, both with MLLr as well as in leukemias carrying mutations in NPM1. Inhibition of the MLL-Menin interaction with novel Menin inhibitors has shown efficacy in both AML subsets, in pre- clinical and clinical studies. However, the responses are short-lived and hence it is imperative to identify therapeutic approaches that may synergize with Menin inhibition. Our recent work showed that MLLr t(4;11) Acute Lymphoblastic leukemia () patient cells express high levels of the anti-apoptotic BCL-2 protein and are highly sensitive to treatment with the BCL-2 selective antagonist, venetoclax (Benito, J.M., et al , Cell Reports, 2015). Here, we report experiments to establish the efficacy of the novel Menin-MLL inhibitor DS-1594b which was developed by Daiichi Sankyo co., jp, in combination with the BCL-2 antagonist venetoclax (VEN) in MLLr and NPM1-mutated acute leukemias. Methods: The efficacy of DS-1594b/VEN combination in MLLr and NPM1mut AML cell lines and primary leukemia samples was evaluated by examining the cell viability in response to serial dilutions of tested compounds as single agents or in combination using Cell Titer Glo (CTG) assay at 3, 5 and 7 days of treatment. Induction of apoptosis was assessed by Annexin V/ staining. Since down-regulation of MLL fusion target genes leads to differentiation in MLL leukemic cells, we monitored cell differentiation by flow cytometry in AML cell lines (MLLr: OCI-AML2, MOLM13, MOLM14, MV411. NPM1 Mut: OCI-AML3 . Leukemia cell lines with no MLLr or NPM1 mutations: HL60, U937) and primary leukemia samples ( blood from relapsed MLLr and NPM1mut AML) using differentiation markers CD11b, CD14, CD15. MLLr cell line MOLM13 resistant to venetoclax was also included in these studies. Results: 7 days treatment with DS-1594b reduced viability in MLLr and NPM1mut cell lines at low nM concentrations but did not affect growth of negative control U937 and HL60 . DS-1594b induced differentiation in OCI-AML3 and Ven resistant cell after 5 days and in other lines after 10 days (figure 1A). DS-1594b did not induce apoptosis when used as a single agent in any AML cell line tested indicating that differentiation is a key mechanism of Menin inhibition. Five days of treatment with single agent showed a downregulation of Menin in all cell lines and downregulation of BCL2 in OCI-AML3 and OCI-AML2 in Western blot analysis. Remarkably, combination with Venetoclax induced synergistic lethality and growth inhibition in all cell lines. Most importantly, was observed in primary leukemia samples (Figure 1B), with differentiation effects after DS-1594b single agent (bottom). Conclusions: Here we show in vitro efficacy of DS-1594b, a selective small molecule menin inhibitor, as single agent or in combination with Venetoclax in MLLr and NPM1 mutated cell lines and primary leukemia samples. Preliminary data demonstrate differentiation effects of DS-1594b as single agent in both cell lines and primary AML leukemia samples. Combination effects show synthetic lethality in almost all cell lines except OCI-AML3. The combination elicited profound apoptosis in primary leukemia samples. In vivo studies using MLLr and NPM1 PDXs models are ongoing and will be presented. Overall, the preliminary findings presented here pave the way for further studies aimed at understanding mechanisms of and further development and testing of this combination in AML with MLLr and/or NMP1 mutations. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Entospletinib (ENTO) in Combination with Cytarabine (Ara-C) and Daunorubicin (DNR) in Newly Diagnosed (ND) Adult Patients with NPM1-Mutated and FLT3-ITD Wild-Type Acute Myeloid Leukemia (AML) Is Associated with Good Response and Survival: A Phase 2 Sub-Study of the Beat AML Master Trial

Entospletinib (ENTO) in Combination with Cytarabine (Ara-C) and Daunorubicin (DNR) in Newly Diagnosed (ND) Adult Patients with NPM1-Mutated and FLT3-ITD Wild-Type Acute Myeloid Leukemia (AML) Is Associated with Good Response and Survival: A Phase 2 Sub-Study of the Beat AML Master Trial

A Phase 1b Dose Escalation and Expansion Study of SNDX-5613, Azacitidine (AZA) and Venetoclax (VEN) in Newly Diagnosed, Patients ≥ 60 Years with Untreated NPM1-Mutated/ FLT3-Wild Type AML or KMT2A-Rearranged Acute Myeloid Leukemia (AML)

Background: SNDX-5613 is a potent, orally available, small molecule inhibitor of menin, which binds to MLL1, encoded by the KMT2A gene, and MLL1 fusion proteins. Menin expression leads to aberrant gene transcription through its activation of Homeobox-related genes, leading to maturation arrest and leukemogenesis in KMT2A-rearranged and NPM1-mutated leukemias. Inhibition of menin activity significantly blocks the transcriptional signature and subsequently the proliferative capacity of leukemia cells harboring NPM1 mutations or KMT2A rearrangements. SNDX-5613 and its analogs have demonstrated robust single-agent anti-proliferative activity in NPM1-mutated AML cells and AML patient-derived xenograft models. Currently, SNDX-5613 is being explored as a single agent in a phase 1/2 study in patients with NPM1-mutated or KMT2A-rearranged relapsed/refractory AML, mixed phenotypic acute leukemia and acute lymphoblastic leukemia. The primary objective of this study is to determine the safety and recommended dose of SNDX-5613 combined with AZA and VEN in untreated AML patients age ≥ 60 years who are not candidates for intensive chemotherapy. Study Design and Methods: This study is a sub-study of the BeatAML Master Trial (NCT03013998) in which untreated AML patients age ≥ 60 are assigned an investigational therapy based on cytogenetic and central genomic analysis. Patients must specifically harbor an identifiable NPM1 mutation without a concomitant FLT3 mutation or have a KMT2A rearrangement to be eligible. In this phase 1b study, the primary objective is to determine the safety and recommended dose using a standard 3+3 design based on dose-limiting toxicities with 3 dose levels (DL) of SNDX-5613 to be evaluated in combination with VEN/AZA and an anti-fungal (required during induction): 75mg (DL-1), 113mg (DL1), and 163mg (DL2). In continuation phase with anti-fungals not required, SNDX-5613 will be dosed at 113mg (DL-1), 163mg (DL1), or 226mg (DL2) with appropriate dose reductions added for both SNDX-5613 and VEN if continuing on anti-fungals. The secondary objectives are to assess the overall response rate, measurable residual disease (MRD) status, overall survival, transplant rate and duration of remission. During induction cycles, VEN (D1-28) and AZA (D1-7) are dosed per standard of care every cycle. SNDX-5613 is dosed continuously orally twice daily with dose levels listed above. In patients who achieve a morphologic remission (<5% blasts), SNDX-5613 will be held if absolute neutrophil count (ANC) is < 0.5x109/L and/or platelets (PLT) < 50x109/L. Patients can receive up to 3 induction cycles to achieve a composite complete remission (CRc: CR, CRi, CRh). Patients can receive continuation therapy with AZA, VEN and SNDX-5613 once they achieve CRc and after hematologic recovery. Following the determination of a safe and recommended dose in phase 1b, there will be a dose expansion cohort of 12-20 patients following the same schedule as described above (Figure 1) to further evaluate safety, tolerability and correlative biomarkers. Correlative objectives include assessment of SNDX-5613 pharmacokinetics and its correlation with response and toxicities. Additionally, the study will evaluate the impact of MRD, as measured by central flow cytometry and next-generation sequencing of NPM1 mutations. Additional evaluations and biomarkers of potential interest include menin- KMT2A-specific transcriptomic changes, myeloid differentiation/cell subset changes, mechanisms of resistance, and discovery of baseline features (methylation, mutations, etc.) that can impact outcomes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

LSC17 Score Is Complementary with Minimal Residual Disease to Stratify NPM1-Mutated Acute Myeloid Leukemia: An ALFA Study

Context. A 17-gene Leukemic Stem Cell gene expression signature (LSC17) has been shown to be prognostic in AML (Ng et al., 2016), independently of genetic risk stratification (Bill et al., 2020; Duployez et al., 2019). Whether assessment of LSC17 adds prognostic value on top of measurable residual disease (MRD) has not been explored. Objective. We analyzed the prognostic value of LSC17 in adult AML patients prospectively treated in the intensive chemotherapy ALFA-0702 trial (NCT00932412) in the context of ELN 2017 risk stratification (ELN17) and NPM1 MRD. Methods. The LSC17 score was determined by Nanostring technology (Duployez et al., 2019). NPM1 MRD negativity was defined as a 4-log reduction in peripheral blood NPM1c expression (Balsat et al., 2017; Lesieur et al., 2021). Multiple testing was adjusted for using the Benjamini-hochberg procedure. Results. The study population included 504 AML patients (237 female, 267 male) with a median age of 48 years (range 18-60). One hundred eighty-seven (37%) patients had an NPM1 mutation. ELN17 risk was favorable in 176 (35%), intermediate in 151 (30%) and adverse in 170 (34%) patients. A higher LSC17 score was positively associated with RUNX1 (median difference [md]=0.13, corrected p-value [q]=0.0005) and negatively correlated with NPM1 mutation (md=-0.14, q<0.0001) and double-mutated CEBPA (md=-0.30, q<0.0001). Regarding recurrent cytogenetic lesions, presence of monosomy 7 or del7q (md=0.29, q<0.0001), monosomy 5 or del5q (md=0.25, q=0.0004), monosomy 17, del17p or abnormal 17p (md=0.24, q=0.01), monosomal karyotype (md=0.17, q=0.02) and complex karyotype (md=0.13, q=0.03) were each associated with higher LSC17, whereas normal karyotype was associated with a lower LSC17 score (md=-0.11, q<0.0001) (Figure 1A). In the subgroup of NPM1-mutated AML (n=187), a higher LSC17 score was only correlated with FLT3-ITD (md=0.13, q=0.0002) but not DNMT3A (md=-0.0007, q=1) mutations. Patients were dichotomized into LSC17-low (n=252) and LSC17-high (n=252) groups using the median LSC17 score as cutoff. Overall, 400 (79%) patients achieved complete response (CR) or CR with incomplete platelet recovery (CRp) after one cycle of induction and an additional 31 (6%) after a second course. LSC17-high patients had a lower rate of CR/CRp after one course of induction (69% vs 89% in LSC17-low patients) in univariate (p<0.0001) or multivariate analysis (OR=0.41, p=0.001) considering ELN17 risk, age and white blood cell (WBC) count. LSC17-high patients had lower 3-year overall survival (OS) from CR/CRp (60.6%, 95% CI, 54.2-67.8%) than LSC17-low patients (75.4%, 95% CI, 70.0-81.2%, p=0.0003). In multivariate analysis considering ELN17, age and WBC, LSC17-high population had shorter DFS (HR=1.39, p=0.038) and a trend for shorter OS from CR/CRp (HR=1.40, p=0.069). In univariate analyses, LSC17-high status was associated with a higher cumulative incidence of relapse (p=0.002) without impact on non-relapse mortality (p=0.27). Considering hematopoietic cell transplantation (allo-HCT) as a time-dependent covariate, there was no interaction on OS from CR/CRp between allo-HCT in first CR and LSC17-high status (p=0.84). In NPM1-mutated patients (n=187), the rate of CR/CRp after one induction course was 90% in LSC17-high patients versus 96% in LSC17-low patients (p=0.20). In 123 NPM1-mutated patients in CR after the first induction course and with available MRD, LSC17-high status (n=42) predicted a poorer DFS (HR:2.47, p=0.005) in multivariate analysis independently of age (p=0.55), higher WBC (p=0.84), intermediate ELN17 risk (p=0.97) and MRD positivity (p=0.0002). LSC17-high status also independently predicted poorer OS from CR/CRp (HR:2.73, p=0.007) in a similar multivariate model. Three-year OS from CR was 93.1% (95% CI, 86.8-99.9%) in the 59 (48%) NPM1-mutated LSC17-low patients achieving negative MRD compared to 60.7% (95% CI, 49.8-74.0%) in those (n=64, 52%) with LSC17-high status and/or positive MRD (p=0.0001) (Figure 1B). Conclusion. The LSC17 score complements genetic risk stratification in AML patients treated intensively. Combining LSC17 and MRD assessment identifies a subset of NPM1-mutated AML with excellent long-term prognosis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

An Unusual Extramedullary Presentation of an NPM1-Mutated Acute Myeloid Leukemia (AML) in a Young Adult

Abstract Introduction/Objective Pleural effusion presentation of AML, particularly without leukemic disease, has been rarely described in the literature. This case serves to investigate the value of an identified NPM1 mutation. Methods/Case Report A 25-year-old male with Down syndrome presented with dyspnea. Computerized tomography scan identified a moderate pleural effusion and pelvic lytic bone lesions. A complete blood count was normal. Pleural fluid cytologic evaluation showed a spectrum of immature myeloid precursors, and flow cytometry identified a 23% CD117 positive myeloblast population expressing CD33, CD13, and dim CD45 but lacking CD34 and HLA-DR. Fluorescence in-situ hybridization testing was negative for a PML-RARA fusion. Given the phenotype, molecular testing was initiated and confirmed an NPM1 mutation. Testing revealed additional pathogenic variants in DNMT3A and IDH1 with no FLT3 mutations. Peripheral blood smear and bone marrow evaluations yielded no clear leukemic involvement, and cytogenetics from bone marrow showed 47,XY,+21c[20]. The significance of this myeloid infiltrate was initially not entirely clear with no established leukemic involvement or tumor-forming mass as required for the World Health Organization diagnosis of myeloid sarcoma. However, the finding of the NPM1 mutation lent high confidence for a diagnosis of an extramedullary presentation of acute myeloid leukemia, though the lytic bone lesions were not biopsied to possibly demonstrate a myeloid sarcoma. Results (if a Case Study enter NA) N/A. Conclusion The identified NPM1 mutation was instrumental in diagnosing an unusual presentation of AML where material was limited. Subsequent to this presentation the patient developed more overt leukemic disease at an outside institution, additionally supporting this diagnosis.

Revisiting coexisting chromosomal abnormalities in NPM1-mutated AML in light of the revised ELN 2022 classification

Mutations in the nucleophosmin 1 gene (NPM1) occur frequently in acute myeloid leukemia (AML) and are associated with a favorable prognosis. Applying the new 2022 European LeukemiaNet (ELN) classifier, Angenendt et al tested the prognostic significance of the copresence of NPM1 mutations and adverse-risk cytogenetics among 2426 patients. The authors demonstrate that outcomes for cytogenetic adverse-risk AML are not modulated by the presence or absence of NPM1 mutations, thereby clarifying management for patients.

AML-377 A “Designed” High-Throughput Drug Screening Strategy Identifies Aurora Kinase A Inhibitors as Promising Preclinical Candidates for the Treatment of NPM1-Mutated AML

Nucleophosmin (NPM1)-mutated acute myeloid leukemia (AML) accounts for one-third of adult AML and displays distinctive biological and clinical features. Despite its chemo-sensitivity and a relatively favorable prognosis, this leukemia lacks any specific therapies. Because NPM1 mutant protein is not druggable, targeting its non-oncogene addictions and its specific vulnerabilities can represent an alternative option for identifying a tailored therapy for NPM1-mutated AML. We performed a high-throughput screening (HTS) of different chemical compounds and drug libraries to find drugs or compounds 'synthetically lethal' with NPM1 mutation and/or showing more selective activity in AML with NPM1 mutation. In collaboration with the Screening Unit of Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP; (Berlin, Germany), we performed an HTS of 38720 drugs/compounds in a 384-well format. The assay readout was cellular metabolism/viability by CellTiter Blue assay at 48 hours. The screening was properly designed in 2 steps to increase efficiency, exclude compounds with general toxicity, and identify compounds ('hits') selectively active against the only two available NPM1-mutated AML cell lines, OCI-AML3 and IMS-M2, but not against wild-type controls. Sixty-two hit compounds that met qHTS threshold criteria were rescreened in a dose-response experiment. From this analysis, we obtained 37 compounds able to selectively kill IMS-M2 (IMS-M2 %viability <50 vs. OCI-AML2 %viability >75). We focused on Aurora kinase A inhibitors (AURKAi) because 4 of the selective compounds were directed against this kinase. In the validation assay, 2 compounds (MK-5108 and MK-8745) were confirmed as more active against NPM1-mutated than NPM1-wild-type AML cells by either CyQUANT or apoptosis assay. Strikingly, we confirmed selectivity for two additional AURKAi (Alisertib and ENMD-2076, both isoform-selective inhibitors of AURKA) that were not included in the screening library. The AURKAi were also effective in reducing tumor burden in NPM1-mutated PDX cell models and provided a survival advantage compared with vehicle-treated mice (60 vs. 32 days, P=0.005). We demonstrated that using HTS can considerably shorten drug discovery time scales, leading to the identification of promising lead compounds. Our study is the first to provide preclinical evidence of the antileukemic activity of AURKAi, with particular focus on NPM1-mutated AML.

Targeted therapy in NPM1-mutated AML: Knowns and unknowns.

Acute myeloid leukemia (AML) is a heterogeneous disease characterized by malignant proliferation of myeloid hematopoietic stem/progenitor cells. NPM1 represents the most frequently mutated gene in AML and approximately 30% of AML cases carry NPM1 mutations. Mutated NPM1 result in the cytoplasmic localization of NPM1 (NPM1c). NPM1c interacts with other proteins to block myeloid differentiation, promote cell proliferation and impair DNA damage repair. NPM1 is a good prognostic marker, but some patients ultimately relapse or fail to respond to therapy. It is urgent for us to find optimal therapies for NPM1-mutated AML. Efficacy of multiple drugs is under investigation in NPM1-mutated AML, and several clinical trials have been registered. In this review, we summarize the present knowledge of therapy and focus on the possible therapeutic interventions for NPM1-mutated AML.

Contemporary outcomes in IDH-mutated acute myeloid leukemia: The impact of co-occurring NPM1 mutations and venetoclax-based treatment.

Isocitrate dehydrogenase 1 or 2 (IDH1 or IDH2) mutations occur frequently in newly diagnosed (ND) acute myeloid leukemia (AML) often with co-occurring NPM1 mutations, which may influence treatment outcomes. Detailed analysis of IDH-mutated AML treated with venetoclax and influence of co-occurring NPM1 mutations remains unclear. This retrospective single-center cohort study evaluated clinical and molecular demographics,response and survival, and impact of co-occurring NPM1 mutations in patients with IDH1 or IDH2-mutated AML. 556 patients with IDH1, IDH2, and/or NPM1 mutated AML were included. Patients with IDH1mut AML (N= 119) were more likely to have older age, sAML, ELN-adverse risk disease, and adverse-risk cytogenetics compared to those with IDH2mut (N= 229) or IDHwt /NPM1mut AML (N= 208). In multivariate analysis, patients with IDH2mut (HR 0.61 [95%CI: 0.43-0.88], p value: .007) or IDHwt /NPM1mut (HR 0.65 [95% CI: 0.45-0.94], p value: .024) AML had a decreased risk of death versus IDH1mut AML. Venetoclax-based lower-intensity regimens partially abrogated the detrimental effect of IDH1mut with similar OS observed between IDH1mut /NPM1wt , IDH2mut /NPM1wt , and IDHwt /NPM1mut AML. With regards to the influence of IDHmut /NPM1mut cases, IC improved survival in IDH2mut /NPM1mut versus IDH2mut /NPM1wt AML (HR: 0.54 [95% CI: 0.2644-1.082], p value: .077), while venetoclax-based therapy improved survival in IDH1mut /NPM1mut versus IDH1mut /NPM1wt AML (HR: 0.094 [95% CI: 0.01-0.74], p value: .0056). Differing outcomes were observed in IDH1mut versus IDH2mut or NPM1mut AML which were influenced by co-occurring NPM1 mutations and partially abrogated with venetoclax-based therapy. Given the differing biology and survival in IDH1mut AML, investigations incorporating molecularly targeted therapies such as IDH inhibitors remain warranted in this subgroup.

Small-Molecule Inhibition of the Acyl-Lysine Reader ENL as a Strategy against Acute Myeloid Leukemia.

AML is a poor-prognosis disease for which new therapeutic approaches are desperately needed. We developed an orally bioavailable inhibitor of ENL, demonstrated its potent efficacy in MLL-rearranged and NPM1-mutated AML, and determined its mechanisms of action. These biological and chemical insights will facilitate both basic research and clinical translation. This article is highlighted in the In This Issue feature, p. 2483.

Prolonged XPO1 inhibition is essential for optimal antileukemic activity in NPM1-mutated AML

AbstractNPM1 is the most frequently mutated gene in adults with acute myeloid leukemia (AML). The interaction between mutant NPM1 (NPM1c) and exportin-1 (XPO1) causes aberrant cytoplasmic dislocation of NPM1c and promotes the high expression of homeobox (HOX) genes, which is critical for maintaining the leukemic state of NPM1-mutated cells. Although there is a rationale for using XPO1 inhibitors in NPM1-mutated AML, selinexor administered once or twice per week did not translate into clinical benefit in patients with NPM1 mutations. Here, we show that this dosing strategy results in only a temporary disruption of the XPO1-NPM1c interaction, limiting the efficacy of selinexor. Because the second-generation XPO1 inhibitor eltanexor can be administered more frequently, we tested the antileukemic activity of prolonged XPO1 inhibition in NPM1-mutated AML models. Eltanexor caused irreversible HOX downregulation, induced terminal AML differentiation, and prolonged the survival of leukemic mice. This study provides essential information for the appropriate design of clinical trials with XPO1 inhibitors in NPM1-mutated AML.

NPM1-Mutated Patient-Derived AML Cells Are More Vulnerable to Rac1 Inhibition.

The prognosis of acute myeloid leukemia (AML) is poor, especially for the elderly population. Targeted therapy with small molecules may be a potential strategy to overcome chemoresistance and improve survival in AML. We investigated the inhibition of the signaling molecule ras-related C3 botulinum toxin substrate 1 (Rac1) in leukemia cells derived from 79 consecutive AML patients, using five Rac1 inhibitors: ZINC69391, ITX3, EHOP-016, 1A-116, and NSC23766. In vitro cell proliferation and apoptosis assays and the assessment of cytokine profiles in culture media were conducted. All five inhibitors had an antiproliferative effect; IC50 ranged from 3–24 µM. They induced significant apoptosis and necrosis compared to the untreated controls (p < 0.0001) at concentrations around IC40 and IC80. A high versus an intermediate or low antiproliferative effect was more common in NPM1-mutated (p = 0.002) and CD34-negative (p = 0.008) samples, and when NPM1 and FLT3 (p = 0.027) were combined. Presence of NPM1 mutation was associated with reduced viability after treatment with EHOP-016 (p = 0.014), ITX3 (p = 0.047), and NSC23766 (p = 0.003). Several cytokines crucial for leukemogenesis were reduced after culture, with the strongest effects observed for 1A-116 and NSC23766. Our findings suggest potent effects of Rac1 inhibition in primary AML cells and, interestingly, samples harboring NPM1 mutation seem more vulnerable.

S130: NPM1 MUTATED AML: IMPACT OF CO-MUTATIONAL PATTERNS - RESULTS OF THE EUROPEAN HARMONY ALLIANCE

Background: Acute myeloid leukemia (AML) is a heterogeneous disease in terms of clinical features, outcomes and genetics. While mutations of NPM1 are usually considered as a favorable prognostic marker, the vast majority of the patients carry several co-mutations that might influence the prognosis. Therefore, a better understanding of the NPM1mut AML mutational landscape is warranted. The large cohort of AML patients collected within the European HARMONY Alliance provides an excellent basis for this purpose. Aims: To identify clinically significant co-mutational patterns in NPM1mut AML in order to establish a revised risk stratification model. Methods: From the HARMONY Alliance AML database, a total of 1001 NPM1mut intensively treated patients were selected. Clinically significant co-mutations were evaluated using graphical patterns created with the Gephi tool and confirmed by detailed survival analysis using Kaplan-Meier and Cox regression models. Finally, a novel multi-state risk stratification model for NPM1mut AML was established. Results: The study population of 1001 NPM1mut AML patients included 57% females and median age was 53 years. Regarding ELN2017 classification, 68% of patients were classified into the favorable, 29% intermediate and 3% adverse risk groups. The most frequent co-mutations were DNMT3A (54%), followed by FLT3-ITD (38%). In total, 24% of patients presented with a high allelic mutant-to-wildtype ratio ≥0.5 (FLT3-ITDhigh) while 14% had low allelic ratio <0.5 (FLT3-ITDlow). Other frequent co-mutations were NRAS (21%), TET2 (20%) and PTPN11 (15%). The triple mutation pattern of NPM1mut + FLT3-ITDhigh + DNMT3Amut identified a subgroup with adverse prognosis (2-year OS of 25%), similar to NPM1mut + TP53mut. The combination of FLT3-ITDlow + DNMT3Amut or FLT3-ITDhigh + DNMT3Awt was associated with intermediate prognosis (2-year OS of 45% and 53% respectively). Notably, mutations of NRAS, KRAS, PTPN11 or RAD21 were identified to be associated with better OS. However, in the context of NPM1mut + DNMT3Amut these mutations did not affect the prognosis when a FLT3-ITD was present. This information is summarized in a 3-category risk classification model (Figure 1). The revised NPM1mut favorable group presented with a 2-year OS of 73%, while for intermediate and adverse groups the OS was 54% and 27% respectively (p<0.001). Regarding relapse free survival (RFS), the median was not reached in the favorable group, while it was 23 months for intermediate and 6 months for adverse group (p<0.001). It should be noted that 171 patients in the NPM1mut intermediate group would be considered as favorable according to the ELN2017 criteria, as well as 162 patients in the NPM1mut adverse group were previously classified as intermediate risk. Therefore, our model was able to reclassify 33% of NPM1mut AML patients in comparison to ELN2017 criteria. Multivariate analysis of OS in NPM1mut AML identified the following independent prognostic factors: NPM1mut model (taking favorable group as reference, HR 1.6 for intermediate and HR 2.7 for adverse group, p<0.001); secondary or therapy-related AML (HR 1.8, p<0.001), WBC at diagnosis >100x103/μL (HR 1.5, p<0.001) and age >60 years (HR 1.4, p<0.001). Image:Summary/Conclusion: Analysis of large NPM1mut AML cohorts allows the discovery of co-mutation patterns associated with prognostic outcome. In accordance, we propose a new genetic stratification model for NPM1mut AML that identifies 3 groups with different OS and RFS. This model improves ELN2017 criteria as it is able to correctly reclassify 33% of NPM1mut AML patients.

P392: PHARMACOLOGICAL INHIBITION OF SYK CONFERS ANTI-PROLIFERATIVE AND NOVEL ANTI-TUMOR IMMUNE RESPONSES IN AML

Background: Spleen tyrosine kinase (SYK) acts as a key integrator of signals from cell surface receptors containing an immunoreceptor tyrosine-based activation motif to boost cellular proliferation. In acute myeloid leukemia (AML), SYK serves as a relay to an oncogenic transcriptional regulatory network linked to NPM1, HOXA9 and MEIS1. The selective, orally bioavailable SYK inhibitor entospletinib (ENTO) has demonstrated clinical activity and tolerability in HOXA9/MEIS1-driven AML. ENTO is currently being investigated in a global Phase 3 trial, AGILITY (NCT05020665), in combination with intensive induction/consolidation chemotherapy in patients (pts) with treatment-naive NPM1-mutated (NPM1m) AML. Lanraplenib (LANRA) is a next-generation SYK inhibitor with similar potency and selectivity to ENTO but with more favorable pharmacologic properties that is currently being evaluated in combination with gilteritinib in pts with relapsed or refractory FLT3-mutated AML (NCT05028751). Aims: To investigate the effects of ENTO and LANRA in T-cell responses in AML. Methods: All pt-derived bone marrow (BM) and peripheral blood samples were obtained in accordance with IRB (OHSU#004422) approval. Results from gene expression profiling studies (RNA sequence [RNA-seq]) in bone marrow/peripheral blood mononuclear cells (MNCs) derived from 152 pts with AML, for which matching NPM1 status and ex vivo sensitivity to ENTO was available, previously published by Tyner et al (2018), were reanalyzed using trimmed mean of M values normalization and differential expression using edgeR. ENTO/LANRA sensitivity was assessed ex vivo after 72 hours in culture at 37°C by MTS assay. T-cell functional assays were performed by culturing MNCs with anti-CD3 and treating with ENTO or LANRA, singly and in combination with anti-PD1. CD3+ T-cell proliferation as measured by Ki67 expression and pSYK activation were assessed by quantitative single-cell immunofluorescence microscopy. Six formalin-fixed, paraffin-embedded BM biopsies from newly diagnosed AML pts were assessed for spatial expression of mRNA using a digital spatial profiling method. Results: In AML, mutations in NPM1 with co-expression of HOXA9/MEIS1 at baseline predicted anti-proliferative activity to ENTO in ex vivo drug sensitivity studies. Accordingly, treatment of leukemic cells with either ENTO or LANRA inhibited SYK auto-phosphorylation in a dose-dependent manner. Pathway analysis of archival RNA-seq data from pts enrolled in the Leukemia and Lymphoma Society BEAT AML Master Protocol (NCT03013998 [BAML-16-001-S6]) revealed gene expression signatures at baseline associated with the observed sensitivity to ENTO, including significant enrichment in the expression of genes associated with leukemogenesis, myeloid differentiation and immune regulation. This was supported by T-cell functional studies, which demonstrated that ENTO and LANRA, singly as well as in combination with anti-PD1, could restore T-cell proliferation in primary AML pt samples that exhibited suppression. Lastly, Cancer Transcriptome Atlas analysis on archival BM biopsies obtained from these AML pts showed a strong correlation between immune checkpoint response and gene expression associated with immune pathway signaling. Summary/Conclusion: Inhibition of SYK is a promising therapeutic approach in NPM1m AML. Our studies illustrate that ENTO and LANRA may restore T-cell proliferation in a subset of AML pts with dysfunctional T-cell responses, suggesting a novel mechanism of action. Additional studies are required to fully understand the mechanism of SYK-mediated antitumor immune responses in AML.

Acute promyelocytic leukemia: Immunophenotype and differential diagnosis by flow cytometry.

Prompt diagnosis of acute promyelocytic leukemia (APL) is critical for patient care. In this study, we aimed to characterize the immunophenotype of APL and explore immunophenotypic difference between APL and its mimics using flow cytometric analysis. Eighty-five cases were collected, including 47 APL, 26 NPM1-mutated acute myeloid leukemia (AML) and 12 KMT2A-rearranged AML with an APL-like immunophenotype. Immunophenotypes were analyzed using flow cytometric analysis. APL showed four distinct patterns (designated a-d) based on CD45/SSC plots. Blasts in patterns a-c showed high side scatter, whereas blasts in pattern d had low side scatter and were located in the traditional blast gate. Compared with patterns a-c, pattern d of APL (APL-D) was more often positive for CD2 (p=0.0005) and CD34 (p=0.0002) in blasts. All NPM1-mutated AML and KMT2A-rearranged AML cases with an APL-like immunophenotype had blasts in the traditional blast gate on CD45/SSC, mimicking APL-D. In comparison, uniform CD13 and positive CD64 were seen in 100% (n=13) APL-D cases and in only 2 of 26 (8%) NPM1-mutated AML cases (p < 0.0001). In addition, APL-D cases were more likely to be positive for CD2 and/or CD34 than NPM1-mutated AML (p <0.0001 and p=0.0007, respectively). In comparison with APL-D, KMT2A-rearranged AML cases were less often positive for myeloperoxidase (MPO) (p=0.001), with none being strongly positive. Similar to NPM1-mutated AML and different from APL-D, KMT2A-rearranged AML cases were rarely positive for CD34 and all negative for CD2. APL and its immunophenotypic mimics share some immunophenotypic similarities but can be distinguished by CD2, CD13, CD34, CD64, and MPO.

Abstract CT210: A phase 1 study of BMF-219, a novel oral irreversible menin inhibitor, as single-agent in patients with relapsed/refractory (R/R) acute leukemia (AL), diffuse large B-cell lymphoma (DLBCL), and multiple myeloma (MM)

Abstract Background: Menin, a protein involved in transcriptional regulation, impacting cell cycle control, apoptosis, and DNA damage repair plays a direct role in oncogenic signaling in multiple cancers. Inhibition of menin is a novel approach to cancer treatment. Preclinical data of BMF-219, a highly selective, orally bioavailable small-molecule irreversible inhibitor of menin, show sustained potent abrogation of menin-dependent oncogenic signaling in vitro and in vivo. BMF-219 demonstrated a strong anti-proliferative effect on various menin-dependent acute myeloid leukemia (AML) cell lines, DLBCL lines representing Double/Triple Hit Lymphoma (DHL/THL), Double Expressor Lymphoma (DEL), and MM cell lines harboring diverse mutational backgrounds. BMF-219 also exhibited high potency ex vivo in patient samples from MLL-rearranged and NPM1-mutant AML, THL and MYC-amplified DLBCL, and bone marrow mononuclear cells from treatment-naive and R/R MM. Methods: BF-MNN-101 (NCT05153330) is a prospective, open-label, multi-cohort, non-randomized, multicenter Phase I study evaluating the safety, tolerability, and clinical activity of escalating doses of oral BMF-219 administered once daily in patients with R/R AL, DLBCL and MM who have received standard therapy. Utilizing an accelerated titration design, doses of BMF-219 will be escalated in single-subject cohorts independently for each indication following a modified Fibonacci sequence until 1 subject experiences either a ≥ Grade 2 adverse event or dose limiting toxicity (DLT). At that point, the current and all subsequent dose level(s) for the specific indication/cohort will follow a classical “3 + 3” design. Each treatment cycle will be 28 days until progression or intolerability occurs. Following conclusion of the parallel dose-escalations, expansion cohorts for each indication will enroll patients to obtain further safety and efficacy data. Eligible patients include those with R/R AL, R/R DLBLC ≥ 2 but ≤ 5 therapies, and R/R MM who received ≥ 3 therapies who failed or are ineligible for any standard therapies. Patients must have ECOG ≤2, and adequate organ function with prior treatment-related toxicities resolved to ≤ Grade 2. Key exclusion criteria include known CNS disease involvement, prior menin inhibitor therapy, and clinically significant cardiovascular disease. The primary objective is to determine independently for each cohort/indication the optimal biological dose (OBD)/recommended Phase 2 dose (RP2D) of BMF-219 oral monotherapy. Key secondary objectives include further evaluation of safety and tolerability, characterization of the pharmacodynamics and pharmacokinetics of BMF-219, and assessment of its antitumor activity based on best overall response rate (ORR), duration of response (DOR), progression-free survival (PFS), time to progression (TTP) per disease-specific response criteria as assessed by the investigator. Food-effect studies will be performed in DLBCL and MM patients at certain dose levels in the escalation phase and during the expansion cohort portion of the study. The first patient in the study is anticipated to dose in Q1 of 2022. Citation Format: Farhad Ravandi-Kashani, Ashwin Kishtagari, Hetty Carraway, Emily Curran, Gary Schiller, Alex Cacovean, Bhagyashree Yadav, Thomas Butler, Jeffrey Lancet. A phase 1 study of BMF-219, a novel oral irreversible menin inhibitor, as single-agent in patients with relapsed/refractory (R/R) acute leukemia (AL), diffuse large B-cell lymphoma (DLBCL), and multiple myeloma (MM) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT210.

Memory-like NK cells armed with a neoepitope-specific CAR exhibit potent activity against NPM1 mutated acute myeloid leukemia

Acute myeloid leukemia (AML) remains a therapeutic challenge, and a paucity of tumor-specific targets has significantly hampered the development of effective immune-based therapies. Recent paradigm-changing studies have shown that natural killer (NK) cells exhibit innate memory upon brief activation with IL-12 and IL-18, leading to cytokine-induced memory-like (CIML) NK cell differentiation. CIML NK cells have enhanced antitumor activity and have shown promising results in early phase clinical trials in patients with relapsed/refractory AML. Here, we show that arming CIML NK cells with a neoepitope-specific chimeric antigen receptor (CAR) significantly enhances their antitumor responses to nucleophosphmin-1 (NPM1)-mutated AML while avoiding off-target toxicity. CIML NK cells differentiated from peripheral blood NK cells were efficiently transduced to express a TCR-like CAR that specifically recognizes a neoepitope derived from the cytosolic oncogenic NPM1-mutated protein presented by HLA-A2. These CAR CIML NK cells displayed enhanced activity against NPM1-mutated AML cell lines and patient-derived leukemic blast cells. CAR CIML NK cells persisted invivo and significantly improved AML outcomes in xenograft models. Single-cell RNA sequencing and mass cytometry analyses identified up-regulation of cell proliferation, protein folding, immune responses, and major metabolic pathways in CAR-transduced CIML NK cells, resulting in tumor-specific, CAR-dependent activation and function in response to AML target cells. Thus, efficient arming of CIML NK cells with an NPM1-mutation-specific TCR-like CAR substantially improves their innate antitumor responses against an otherwise intracellular mutant protein. These preclinical findings justify evaluating this approach in clinical trials in HLA-A2+ AML patients with NPM1c mutations.

COVALENT-101: A phase 1 study of BMF-219, a novel oral irreversible menin inhibitor, in patients with relapsed/refractory (R/R) acute leukemia (AL), diffuse large B-cell lymphoma (DLBCL), and multiple myeloma (MM).

TPS7064 Background: Menin, a protein involved in transcriptional regulation, impacting cell cycle control, apoptosis, and DNA damage repair, plays a direct role in oncogenic signaling in multiple cancers. Inhibition of menin is therefore a novel approach to cancer treatment. Preclinical data of BMF-219, a highly selective, orally bioavailable, small-molecule irreversible inhibitor of menin, show sustained potent abrogation of menin-dependent oncogenic signaling in vitro and in vivo. BMF-219 exhibited a strong anti-proliferative effect on various menin-dependent acute myeloid leukemia (AML) cell lines, DLBCL lines representing Double/Triple Hit Lymphoma (DHL/THL) and Double Expressor Lymphoma (DEL), and MM cell lines with diverse mutational backgrounds. BMF-219 also showed high potency ex vivo in patient samples from MLL-rearranged and NPM1-mutant AML, THL and MYC-amplified DLBCL, and bone marrow mononuclear cells from treatment-naive and R/R MM. Methods: COVALENT-101 (NCT05153330) is an open-label, multi-cohort, non-randomized, multicenter Phase I study evaluating the safety, tolerability, and clinical activity of escalating doses of once daily oral BMF-219 in patients with R/R AL, DLBCL and MM who have received standard therapy. Utilizing an accelerated titration design, doses of BMF-219 will be escalated in single-subject cohorts independently for each indication until 1 subject experiences either a ≥ Grade 2 related-adverse event or dose limiting toxicity (DLT). At that point, the cohort will switch to a classical “3 + 3” design. Treatment will continue in 28-day cycles until progression or intolerability. Expansion cohorts for each indication will enroll patients to obtain further safety and efficacy data. Patients with R/R AL, R/R DLBCL ≥ 2 but ≤ 5 therapies, and R/R MM who received ≥ 3 therapies and failed or are ineligible for any standard therapies are eligible. Patients must have ECOG PS ≤ 2, and adequate organ function. Key exclusion criteria include known CNS disease involvement, prior menin inhibitor therapy, and clinically significant cardiovascular disease. The primary objective is to determine independently for each cohort/indication the optimal biological dose (OBD)/ recommended Phase 2 dose (RP2D) of BMF-219 oral monotherapy. Key secondary objectives include further evaluation of safety and tolerability, characterization of the pharmacodynamics and pharmacokinetics of BMF-219, and assessment of its antitumor activity based on best overall response rate (ORR), duration of response (DOR), progression-free survival (PFS), and time to progression (TTP) per disease-specific response criteria as assessed by the investigator. Food-effect studies will be performed in DLBCL and MM patients at certain dose levels. The enrollment commenced in January 2022. Clinical trial information: NCT05153330.

PTPN11 mutations in adult acute myeloid leukaemia: Prevalence and clinical implications in the context of NPM1 mutation

More than 90% of NPM1-mutated acute myeloid leukaemia (NPM1mut AML) patients have been determined to harbour other known concurrent mutations. However, there is limited data on the frequency of PTPN11 and its clinical impact in NPM1mut AML. Next-generation sequencing(NGS) was performed retrospectively on 112 genes in 254 patients with NPM1mut AML. Overall, 33 PTPN11 mutations were detected in 29 of the 254 patients (11.42%) screened. PTPN11 alterations were exclusively missense mutations affecting residues located within the N-SH2 (n = 25) and PTP (n = 8) domains and clustered overwhelmingly in exon 3 (n = 25). NPM1mut AML patients with mutant PTPN11 had significant associations with mutations in epigenetic regulators(TET2, IDH1/2, and DNMT3A) (72.41% vs. 46.67%, P = 0.009) and cohesins (RAD21,SMC1A, and SMC3)(10.34% vs. 1.33%, P = 0.02) compared to patients with wild-type PTPN11. Among the patients treated with intensive chemotherapy, the differences in disease-free survival (DFS) and overall survival (OS) between those with mutant and wild-type PTPN11 were not significant (P = 0.4, 0.83, respectively). In conclusion, PTPN11 mutations were frequently identified in NPM1mut AML patients and clustered in exon 3. PTPN11 mutations more frequently co-occurred with mutations involving epigenetic regulators but had no impact on prognosis.