Nucleophosmin Mutations Research Articles

Molecular characteristics of NPM1 mutations in AML patients using targeted NGS

ABSTRACT Background Acute myeloid leukemia (AML) is a clonal hematopoietic stem cell malignancy characterized by the accumulation of myeloid progenitor cells with arrested differentiation, leading to the suppression of normal hematopoiesis. The disease exhibits significant heterogeneity, with less than 30% five-year overall survival. Nucleophosmin 1 (NPM1) is the most commonly mutated gene in adult AML, found in approximately 25–35% of patients, and is recognized as a distinct subtype by the WHO. Methods The mutational status of NPM1 was assessed using the Oncomine Myeloid research panel on the Ion5S NGS platform (Thermo Fisher Scientific, USA). Results In our AML cohort, NPM1 was the most frequently mutated gene (75%). Indel mutations in the NPM1 gene, frequently resulting in frameshift consequences, show a statistically significant difference in variant allele frequency (VAF) compared to other mutation types. Specifically, NPM1-mutated cases exhibit a VAF that is positively correlated with initial bone marrow (BM) blast counts. Univariate logistic regression analysis reveals that a high VAF is significantly associated with non-complete remission (Non-CR), while a low VAF is significantly associated with complete remission (CR), indicating a statistically significant difference in outcomes (p = 0.012). Conclusion Targeted NGS technology enables the simultaneous analysis of multiple AML-relevant genes, offering a comprehensive mutational profile that supports risk stratification, diagnosis, and personalized treatment. Our findings highlight the significant impact of NPM1 mutations on patient outcomes, revealing their molecular characteristics and their correlation with different clinical parameters.

Recent advances in AML with mutated NPM1.

Nucleophosmin 1 (NPM1) mutation is one of the most prevalent genetic mutations in adult acute myeloid leukemia (AML) and is particularly predominant in AML with a normal karyotype. NPM1 is a chaperone protein that plays various roles in several cellular processes. Wild-type NPM1 is normally localized to the nucleus, whereas mutant NPM1 proteins exhibit altered cytoplasmic localization. Clinically, AML with mutated NPM1 without FLT3-ITD is associated with a higher complete remission rate and improved overall survival. AML with mutated NPM1 is categorized as a distinct genetic entity in the World Health Organization classification of hematopoietic malignancies due to its unique clinical and biological features. However, the precise roles of NPM1 in normal hematopoiesis and in AML development remain unclear. Recent studies have revealed various clinical applications of NPM1 mutations in AML treatment, particularly in measurable residual disease analyses that target mutant NPM1 transcripts and in potential therapeutic applications of menin inhibitors and XPO-1 inhibitors for AML with mutated NPM1. Thus, NPM1 mutation is highly significant in AML classification, prognosis, response assessment, and molecular targeted therapies. Here, we review recent progress in clinical and biological aspects of AML with mutated NPM1 including molecular targeted therapy.

Evolution from an antecedent chronic myeloid malignancy does not impact survival outcomes in NPM1-mutated AML.

Nucleophosmin-1 (NPM1)-mutated AML is a molecularly defined subtype typically associated with favorable treatment response and prognosis; however, its prognostic significance in AML evolving from an antecedent chronic myeloid malignancy is unknown. This study's primary objective was to determine the impact of mutated NPM1 on the prognosis of AML evolving from an antecedent chronic myeloid malignancy. We conducted a retrospective chart review including patients with NPM1-mutated de novo and sAML. sAML was defined as those with a preceding chronic-phase myeloid malignancy before diagnosis of AML. Of 575 NPM1-mutated patients eligible for inclusion in our study, 51 (8.9%) patients were considered to have sAML. The median time from diagnosis of NPM1-mutated chronic myeloid malignancy to sAML evolution was 3.6 months (0.5-79.3 months). No significant differences in leukemia-free (2-year LKFS 52.0% vs. 51.2%, p = .9922) or overall survival (2-year OS 56.3% vs. 49.4%, p = .4246) were observed between patients with NPM1-mutated de novo versus sAML. Our study suggests that evolution from a preceding myeloid malignancy is not a significant predictor of poor prognosis in the setting of an NPM1 mutation. Our study demonstrated a short time to progression to sAML in most patients, which further supports the consideration of NPM1 as an AML-defining mutation.

Evaluation of a New Closed-System Automated RT-qPCR Assay for the Rapid Detection and Monitoring of Common Nucleophosmin Mutations in Patients with Acute Myeloid Leukemia.

Quantitative assessment of nucleophosmin 1 (NPM1) mutation status is integral to evaluating measurable residual disease (MRD) in NPM1-mutated acute myeloid leukemia (AML) patients. In a retrospective study, leftover peripheral blood (PB) specimens (n = 40) which were collected for routine clinical diagnostic evaluations of AML disease burden were tested by both a novel automated RT-qPCR quantitative NPM1 assay (Xpert NPM1 mutation assay) and the NPM1 mutA, mutB&D MutaQuant kit. Based on a Deming regression analysis, there was a high correlation (slope = 0.92; intercept = 0.12; Pearson's r = 0.982) between the quantitative results of the Xpert NPM1 mutation assay and the NPM1 mutA, mutB&D MutaQuant kit. The Xpert test quantitative results are thus highly correlated with the comparator method and the former has potential as a useful alternative for the monitoring of AML patients with a known NPM1 mutation.

Cytoplasmic TP53INP2 acts as an apoptosis partner in TRAIL treatment: the synergistic effect of TRAIL with venetoclax in TP53INP2-positive acute myeloid leukemia

BackgroundAcute myeloid leukemia (AML) is a hematopoietic malignancy with poor outcomes, especially in older AML patients. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered a promising anticancer drug because it selectively induces the extrinsic apoptosis of tumor cells without affecting normal cells. However, clinical trials have shown that the responses of patients to TRAIL are significantly heterogeneous. It is necessary to explore predictable biomarkers for the preselection of AML patients with better responsiveness to TRAIL. Here, we investigated the critical role of tumor protein p53 inducible nuclear protein 2 (TP53INP2) in the AML cell response to TRAIL treatment.MethodsFirst, the relationship between TP53INP2 and the sensitivity of AML cells to TRAIL was determined by bioinformatics analysis of Cancer Cell Line Encyclopedia datasets, Cell Counting Kit-8 assays, flow cytometry (FCM) and cell line-derived xenograft (CDX) mouse models. Second, the mechanisms by which TP53INP2 participates in the response to TRAIL were analyzed by Western blot, ubiquitination, coimmunoprecipitation and immunofluorescence assays. Finally, the effect of TRAIL alone or in combination with the BCL-2 inhibitor venetoclax (VEN) on cell survival was explored using colony formation and FCM assays, and the effect on leukemogenesis was further investigated in a patient-derived xenograft (PDX) mouse model.ResultsAML cells with high TP53INP2 expression were more sensitive to TRAIL in vitro and in vivo. Gain- and loss-of-function studies demonstrated that TP53INP2 significantly enhanced TRAIL-induced apoptosis, especially in AML cells with nucleophosmin 1 (NPM1) mutations. Mechanistically, cytoplasmic TP53INP2 maintained by mutant NPM1 functions as a scaffold bridging the ubiquitin ligase TRAF6 to caspase-8 (CASP 8), thereby promoting the ubiquitination and activation of the CASP 8 pathway. More importantly, simultaneously stimulating extrinsic and intrinsic apoptosis signaling pathways with TRAIL and VEN showed strong synergistic antileukemic activity in AML cells with high levels of TP53INP2.ConclusionOur findings revealed that TP53INP2 is a predictor of responsiveness to TRAIL treatment and supported a potentially individualized therapeutic strategy for TP53INP2-positive AML patients.

Clinical prognostic value of different NPM1 mutations in acute myeloid leukemia patients.

Acute myeloid leukemia (AML) patients with various nucleophosmin 1 (NPM1) mutations are controversial in the prognosis. This study aimed to investigate the prognosis of patients according to types of NPM1 mutations (NPM1mut). Bone marrow samples of 528 patients newly diagnosed with AML, were collected for morphology, immunology, cytogenetics, and molecular biology examinations. Gene mutations were detected by next-generation sequencing (NGS) technology. About 25.2% of cases exhibited NPM1mut. 83.5% of cases were type A, while type B and D were respectively account for 2.3% and 3.0%. Furthermore, 15 cases of rare types were identified, of which 2 cases have not been reported. Clinical characteristics were similar between patients with A-type NPM1 mutations (NPM1A - type mut) and non-A-type NPM1 mutations (NPM1non - A-type mut). Event-free survival (EFS) was significantly different between patients with low NPM1non - A-type mut variant allele frequency (VAF) and low NPM1A - type mut VAF (median EFS = 3.9 vs. 8.5 months, P = 0.020). The median overall survival (OS) of the NPM1non - A-type mutFLT3-ITDmut group, the NPM1A - type mutFLT3-ITDmut group, the NPM1non - A-type mutFLT3-ITDwt group, and the NPM1A - type mutFLT3-ITDwt group were 3.9, 10.7, 17.3 and 18.8 months, while the median EFS of the corresponding groups was 1.4, 5.0, 7.6 and 9.2 months (P < 0.0001 and P = 0.004, respectively). No significant difference was observed in OS and EFS between patients with NPM1A - type mut and NPM1non - A-type mut. However, types of NPM1 mutations and the status of FLT3-ITD mutations may jointly have an impact on the prognosis of AML patients.

Monocytic Differentiation of Human Acute Myeloid Leukemia Cells: A Proteomic and Phosphoproteomic Comparison of FAB-M4/M5 Patients with and without Nucleophosmin 1 Mutations.

Even though morphological signs of differentiation have a minimal impact on survival after intensive cytotoxic therapy for acute myeloid leukemia (AML), monocytic AML cell differentiation (i.e., classified as French/American/British (FAB) subtypes M4/M5) is associated with a different responsiveness both to Bcl-2 inhibition (decreased responsiveness) and possibly also bromodomain inhibition (increased responsiveness). FAB-M4/M5 patients are heterogeneous with regard to genetic abnormalities, even though monocytic differentiation is common for patients with Nucleophosmin 1 (NPM1) insertions/mutations; to further study the heterogeneity of FAB-M4/M5 patients we did a proteomic and phosphoproteomic comparison of FAB-M4/M5 patients with (n = 13) and without (n = 12) NPM1 mutations. The proteomic profile of NPM1-mutated FAB-M4/M5 patients was characterized by increased levels of proteins involved in the regulation of endocytosis/vesicle trafficking/organellar communication. In contrast, AML cells without NPM1 mutations were characterized by increased levels of several proteins involved in the regulation of cytoplasmic translation, including a large number of ribosomal proteins. The phosphoproteomic differences between the two groups were less extensive but reflected similar differences. To conclude, even though FAB classification/monocytic differentiation are associated with differences in responsiveness to new targeted therapies (e.g., Bcl-2 inhibition), our results shows that FAB-M4/M5 patients are heterogeneous with regard to important biological characteristics of the leukemic cells.

A 69 long noncoding RNA signature predicts relapse and acts as independent prognostic factor in pediatric AML

AbstractRisk stratification using genetics and minimal residual disease has allowed for an increase in the cure rates of pediatric acute myeloid leukemia (pedAML) to up to 70% in contemporary protocols. Nevertheless, ∼30% of patients still experience relapse, indicating a need to optimize stratification strategies. Recently, long noncoding RNA (lncRNA) expression has been shown to hold prognostic power in multiple cancer types. Here, we aimed at refining relapse prediction in pedAML using lncRNA expression. We built a relapse–related lncRNA prognostic signature, named AMLlnc69, using 871 transcriptomes of patients with pedAML obtained from the Therapeutically Applicable Research to Generate Effective Treatments repository. We identified a 69 lncRNA signature AMLlnc69 that is highly predictive of relapse risk (c-index = 0.73), with area under the receiver operating characteristic curve (AUC) values for predicting the 1-, 2-, and 3-year relapse-free survival (RFS) of 0.78, 0.77, and 0.77, respectively. The internal validation using a bootstrap method (resampling times = 1000) resulted in a c-index of 0.72 and AUC values for predicting the 1-, 2-, and 3-year RFS of 0.77, 0.76, and 0.76, respectively. Through a Cox regression analysis, AMLlnc69, nucleophosmin mutation, and white blood cell at diagnosis were identified as independent predictors of RFS. Finally, a nomogram was build using these 2 parameters, showing a c-index of 0.80 and 0.71 after bootstrapping (n = 1000). In conclusion, the identified AMLlnc69 will, after prospective validation, add important information to guide the management of patients with pedAML. The nomogram is a promising tool for easy stratification of patients into a novel scheme of relapse-risk groups.

Abstract 2113: HMPL-506, a novel, highly potent and differentiated menin-MLL inhibitor for the treatment of MLL-rearrangedand NPM1mutant acute leukemia in preclinical models

Abstract Background: Mixed-lineage leukemia (MLL, or KMT2A) gene rearrangements (MLL-r) occur in 5%-10% acute leukemias and are associated with poor prognosis. Nucleophosmin 1 mutations (NPM1m) are the most common genetic alterations in acute myeloid leukemia (AML). Multiple studies have illuminated that those leukemogenesis are dependent on the interaction of menin-MLL, which controls downstream gene expressions associated with cell proliferation and differentiation, e.g. HOXA9 and CD11b. Herein, we introduce HMPL-506, a novel and highly differentiated small molecule compound targeting menin-MLL interaction. Methods and Results: Biochemical assay revealed that HMPL-506 potently blocked menin-MLL binding with IC50 of 1.0 nM. In cell lines carrying MLL-r or NPM1m, HMPL-506 substantially down-regulated menin-MLL target genes transcription of MEIS1 and HOXA9 and upregulated the differentiation marker of CD11b by RT-PCR assay, consequently, HMPL-506 attenuated tumor cell growth, with GI50 ranging from 3.0 to 12.1 nM in MLL-r cell lines (MV-4-11, MOML-13 and RS4;11) and 22.4 nM in NPM1m cell line (OCI-AML-3) in CellTiter-Glo cell viability assay. Notably, compared with the other 5 menin inhibitors in clinical stage, HMPL-506 showed the strongest inhibitory potency in MLL-r and NPM1m cell line models. In vivo studies demonstrated the dose- and exposure- dependent target regulation of MEIS1 and anti-tumor efficacy following HMPL-506 treatment in multiple tumor xenograft models. For instance, in MV-4-11 subcutaneous model, once daily oral administration of HMPL-506 at 5, 10 and 25 mg/kg for 4 days in BALB/c nude mice induced dose-dependent inhibition of MEIS1 gene expression, positively correlating with compound exposures in tumor tissues; continuous dosing of HMPL-506 at 5 mg/kg for 28 days reduced the tumor growth by 86%, which was comparable to the efficacy induced by SNDX-5613 at 50 mg/kg. Treatment of HMPL-506 at 10 mg/kg and 25 mg/kg resulted in tumor shrinkage in all treated animals, with tumor regression rates of 72% and 100%, respectively. Furthermore, HMPL-506 synergistically improved anti-tumor effect of azacytidine, venetoclax and gilteritinib against MLL-r leukemias both in vitro and in vivo. HMPL-506 displayed favorable PK profiles and high selectivity among multiple kinases, methyltransferases and safety related targets. More importantly, the IC50 of HMPL-506 on hERG patch clamp was over 60 µM, indicating low risk of QTc prolongation in human. Conclusion: HMPL-506 is a novel and highly differentiated menin-MLL inhibitor with robust anti-tumor activities, favorable ADME properties and low risk of cardiac toxicity, warranting it further clinical development for treatment of MLL-r and NPM1m acute leukemias. HMPL-506 IND is expected to be cleared in H1 2024. Citation Format: Min Cheng, Liang Ge, Zhihu Gao, Zeyu Zhong, An Jiang, Wei Zhang, Jia Hu, Shuwen Jiang, Na Li, Na Yang, Jian Wang, Yang Sai, Weiguo Qing, Yongxin Ren, Weiguo Su. HMPL-506, a novel, highly potent and differentiated menin-MLL inhibitor for the treatment of MLL-rearrangedand NPM1mutant acute leukemia in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2113.

Automated manufacture of ΔNPM1 TCR-engineered T cells for AML therapy

Acute myeloid leukemia (AML) is a heterogeneous malignancy that requires further therapeutic improvement, especially for the elderly and for subgroups with poor prognosis. A recently discovered Tcell receptor (TCR) targeting mutant nucleophosmin 1 (ΔNPM1) presents an attractive option for the development of a cancer antigen-targeted cellular therapy. Manufacturing of TCR-modified Tcells, however, is still limited by a complex, time-consuming, and laborious procedure. Therefore, this study specifically addressed the requirements for a scaled manufacture of ΔNPM1-specific Tcells in an automated, closed, and good manufacturing practice-compliant process. Starting from cryopreserved leukapheresis, 2E8 CD8-positive Tcells were enriched, activated, lentivirally transduced, expanded, and finally formulated. By adjusting and optimizing culture conditions, we additionally reduced the manufacturing time from 12 to 8days while still achieving a clinically relevant yield of up to 5.5E9 ΔNPM1 TCR-engineered Tcells. The cellular product mainly consisted of highly viable CD8-positive Tcells with an early memory phenotype. ΔNPM1 TCR CD8 Tcells manufactured with the optimized process showed specific killing of AML invitro and invivo. The process has been implemented in an upcoming phase 1/2 clinical trial for the treatment of NPM1-mutated AML.

Diagnostic utility of immunohistochemistry in detection of NPM1 mutations in acute myeloid leukemia with a patchy distribution.

Nucleophosmin 1 (NPM1) mutations occur in approximately one-third cases of adult de novo acute myeloid leukemia (AML). Identification of NPM1 mutations is important for classification, risk stratification, tailored therapy, and monitoring minimal residual disease. Mutational analysis is widely used for detecting NPM1 mutations. Immunochemistry assessing abnormal cytoplasmic localization of NPM1 protein has been used as a surrogate marker for NPM1 mutations. We present a case of AML with mutated NPM1 that was missed by sequencing analysis but detected by immunohistochemistry. This case highlights the value of immunohistochemistry in identifying NPM1 mutations in a subset of AML cases.

Small Molecule Menin Inhibitors: Novel Therapeutic Agents Targeting Acute Myeloid Leukemia with KMT2A Rearrangement or NPM1 Mutation.

Recent advances have included insights into the clinical value of genomic abnormalities in acute myeloid leukemia (AML) and consequently the development of numerous targeted therapeutic agents that have improved clinical outcome. In this setting, various clinical trials have recently explored novel therapeutic agents either used alone or in combination with intensive chemotherapy or low-intensity treatments. Among them, menin inhibitors could represent a novel group of targeted therapies in AML driven by rearrangement of the lysine methyltransferase2A (KMT2A) gene, previously known as mixed-lineage leukemia (MLL), or by mutation of the nucleophosmin1 (NPM1) gene. Recent phase1/2 clinical trials confirmed the efficacy of SNDX-5613 (revumenib) and KO-539 (ziftomenib) and their acceptable tolerability. Several small molecule menin inhibitors are currently being evaluated as a combination therapy with standard of care treatments. The current paper reviews the recent progress in exploring the inhibitors of menin-KMT2A interactions and their application prospects in the treatment of acute leukemias.

The roles of phosphorylation of signaling proteins in the prognosis of acute myeloid leukemia.

Signaling pathways of Retinoblastoma (Rb) protein, Akt-kinase, and Erk-kinase (extracellular signal-regulated kinase) have an important role in the pathogenesis of acute myeloid leukemia. Constitutive activation of these proteins by phosphorylation contributes to cell survival by regulation of cell cycle, proliferation and proapoptotic signaling processes. According to previous data phosphorylated forms of these proteins represent a worse outcome for cancer patients. We investigated the presence of phosphorylated Rb (P-Rb), Akt (P-Akt) and Erk (P-Erk) proteins by Western blot technique using phospho-specific antibodies in bone marrow or peripheral blood samples of 69 AML patients, 36 patients with myelodysplastic syndrome (MDS) and 10 healthy volunteers. Expression level of PTEN (Phosphatase and tensin homolog) and PHLPP (PH domain and leucine-rich repeat Protein Phosphatase) phosphatases, the negative regulators of Akt kinase pathway were also examined. We tested the effect of these proteins on survival and on the correlation with known prognostic features in AML. We found 46.3% of AML patients had detectable P-Rb, 34.7% had P-Akt and 28.9% had P-Erk protein. 66.1% of patients expressing PTEN, 38.9% PHLPP, 37.2% both PTEN and PHLPP and 32.2% neither PTEN nor PHLPP phosphatases. Compared to nucleophosmin mutation (NPMc) negative samples P-Erk was significantly less in nucleophosmin mutated patients, P-Rb was significantly less in patients' group with more than 30G/L peripheral leukocyte count by diagnosis. PHLPP was significantly present in FAB type M5. The expression of P-Rb represented significant better overall survival (OS), while P-Akt represented significantly worse event-free survival (EFS) in unfavorable cytogenetics patients. The presence of both PHLPP and PTEN phosphatases contributes to better OS and EFS, although the differences were not statistically significant. We confirmed significant positive correlation between P-Akt and PHLPP. Assessing the phosphorylation of Rb, Akt and Erk may define a subgroup of AML patients who would benefit especially from new targeted treatment options complemented the standard chemotherapy, and it may contribute to monitoring remission, relapse or progression of AML.

One-Pot Era-CRISPR/Cas12a System for Rapid and Sensitive Detection of Multiple NPM1 Mutations

Nucleophosmin 1 (NPM1) gene c.863_864 4-bp insertion mutations occur in 30% of adult acute myeloid leukemia (AML) patients, which is associated with a unique pathological phenotype and good prognostic significance. The detection of NPM1 mutations is important for risk stratification, treatment guidance, and minimal residual disease monitoring in AML. However, the current detection methods have their limitations in terms of equipment requirements, sensitivity, turnaround time, price, etc. Moreover, simultaneous detection of multiple NPM1 mutation types is challenging. Here, we developed a one-pot ERA-CRISPR/Cas12a-based assay in which only one guide crRNA is needed to detect hundreds of NPM1 gene variants, covering over 97% of NPM1-mutated AML cases. By optimizing multiple parameters, this assay finally achieved a sensitivity of as low as 0.01% within 30 min detection under constant 39℃, without wild-type related cross signal. In clinical detection, 21 NPM1 mutation positive cases from 70 AML patient were successfully screened out using our method, including two cases not detected by Sanger sequencing. This method is one-step, rapid, sensitive, specific, inexpensive, and without the need of Sequencer or temperature cycler. It holds the potential to assist clinicians in point of care testing.

Myeloid Master Regulators PU.1 and CEBPA Suppress Leukemogenesis through Downregulating Cell Cycle Related Genes in NPM1 Mutated Leukemia

Nucleophosmin1 mutation (NPM1c) is the most common mutation in adult acute myeloid leukemia (AML) and known as a driver mutation causing leukemogenesis through upregulating homeotic gene transcription. Mutated NPM1 is reported to mis-localize in the cytoplasm. However, how cytoplasmic NPM1c regulates leukemic transcription remains poorly understood. We previously reported that NPM1c reprograms homeotic and leukemogenic transcription through reshaping CTCF-defined three-dimensional topology associated domain (TAD). Also, re-localization of cytoplasmic NPM1c to the nucleus switches MIZ1/MYC repressive transcription regulatory axis to MIZ1/NPM1 active transcription regulatory axis at CDKN locus and CEBPA locus leading to cell cycle arrest and myeloid differentiation in NPM1 mutated AML. In this study, we are focused on regulation of myeloid differentiation and hypothesized upregulation of myeloid master regulators, PU.1 and CEBPA may suppress the leukemogenic transcription and induce myeloid differentiation in NPM1 mutated AML cells. To confirm NPM1c regulates TAD formation at CEBPA locus, we performed HiC-seq in OCI-AML3 with nuclear re-localized NPM1c using XPO1 inhibitor treatment and NPM1-wildtype overexpression. Nuclear re-localized NPM1 upregulates CTCF-defined TAD interaction at CEBPA locus and upregulates CEBPA and CEBPD transcription which indicates NPM1c regulates CEBPA transcription via altering CEBPA TAD formation. We then test whether activation of CEBPA or SPI1 in NPM1 C+ AML cells induced myeloid differentiation and blocked NPM1 C+ driven leukemogenesis. We transcriptionally activated SPI1 or CEBPA gene in OCI-AML3 using CRISPR-dCas9-VP160 system to determine how these myeloid regulators changed the leukemic transcription and leukemic cell phenotype. PU.1- or CEBPA- activated OCI-AML3 reduces cell proliferation, especially affecting the G2/M phase in cell cycle. They have monocyte-like nuclei with large cytoplasm and induce myeloid differentiation with upregulating both of CD11b and CD14 myeloid cell surface markers. Moreover, compared to parental OCI-AML3 cells, Pu.1- or CEBPA-activated cells showed lower colony formation ability and in xenograft mouse models produced diminished leukemic symptoms and prolonged survival. We further conducted RNA-seq to explore how PU.1 or CEBPA changed the gene transcription leading to the blockage of leukemogenesis. Between PU.1- and CEBPA-activated cells, 58% of differentially expressed genes overlap and GO or GSEA analysis revealed that both of them downregulates G2M checkpoint/mitotic-spindle cell cycle related gene sets, indicating that PU.1 or CEBPA activation controls cell cycle control rather than myeloid cell differentiation in NPM1 C+ AML. In summary, our data revealed that re-localization of NPM1 C+ into nucleus activates master myeloid transcription factors, PU.1 and CEBPA, which, in turn, suppress leukemogenesis via inducing cell cycle arrest and myeloid cell differentiation in NPM1 C+ AML.

Predicting Molecular Alterations from Blast Morphology of Acute Myeloid Leukemia Bone Marrow Aspirate Smears Using Machine Learning

Introduction Acute Myeloid Leukemia (AML) has enormous molecular and cytogenetic heterogeneity. Evaluation of bone marrow morphology is essential in the diagnosis of AML, however, it requires significant training and there is wide inter-observer variability. Genomic testing has been progressively relied on for AML classification, ultimately helping to dictate prognosis and guide clinical management. However, genomic testing, if performed, can be subject to prolonged turnaround times and lacks clinical penetration in low-resource settings. To overcome these challenges, we aimed to evaluate the feasibility of predicting clinically relevant genomic alterations from bone marrow cell morphology using a machine learning approach. Methods We scanned 799 Wright-stained bone marrow aspirate smear slides, made for routine patient care, for subjects diagnosed with AML. These subjects were linked to clinical data, including pertinent cytogenetic/molecular alterations and subclassified as cases with mutated Nucleophosmin 1 ( NPM1), those with TP53 mutations and those with myelodysplastic-related changes (AML-MRC). Whole slide images were screened for containing spicules and blasts, and were analyzed to detect 180 million cells (blasts and non-blasts) excluding hemodilute and particle regions. We developed a multi-instance model to predict patient molecular subtype by repeatedly sampling 512 cells from each slide and using these to train a classifier that weights cells with morphology relevant to subtype. We also trained a model to predict NPM1 and TP53 mutations independently, as well as other frequent variants reported to impact blast morphology. Results Performance in predicting NPM1/TP53/AML-MRC subtype was moderate (0.80 AUROC). Performance of the single-gene model varied, with NPM1 having the highest performance (0.85 AUROC) followed by IDH (0.80 AUROC), FLT3 (0.79 AUROC) and TP53 (0.62 AUROC). Conclusions This study provides promising preliminary results for prediction of genetic alterations in AML in a large-scale single-institution dataset. Interestingly, this method did not rely on the classification of individual cells, avoiding the need for manually collecting annotations to train a blast cell classifier. Future work will evaluate the morphology of cells attended by the multi-instance model, and explore the value of enriching sampled cells with blasts using cell classification methods.

Revumenib Monotherapy in Patients with Relapsed/Refractory KMT2Ar Acute Leukemia: Topline Efficacy and Safety Results from the Pivotal Augment-101 Phase 2 Study

Background: Most patients (pts) with histone-lysine N-methyltransferase 2A (KMT2A)-rearranged (KMT2Ar) acute leukemia, relapse after conventional chemotherapy and hematopoietic stem cell transplant (HSCT). Remission rates after relapse (complete remission [CR], 5%) and median overall survival (2.4 mo) in ≥2nd salvage therapies in adults remain low (Blood Cancer J. 2021;11[9]:162). In KMT2Ar leukemia, interaction of menin with KMT2A fusion proteins is a key driver of leukemogenesis. However, no therapies targeting the menin-KMT2A interaction have been approved. Revumenib (SNDX-5613; rev), a small-molecule inhibitor of menin-KMT2A interactions, demonstrated preliminary efficacy and safety in a phase 1 study of R/R KMT2Ar and nucleophosmin 1-mutated (NPM1m) acute leukemias. We report topline efficacy and safety for pts with R/R KMT2Ar acute leukemia treated with rev in a pivotal phase 2 study (AUGMENT-101; NCT04065399). Methods: Pts aged ≥30 days with R/R KMT2Ar acute leukemia were enrolled in cohort A (acute lymphoblastic leukemia [ALL]/mixed phenotype acute leukemia [MPAL]) and B (acute myeloid leukemia [AML]); cohort C continues to enroll pts with NPM1m and is not included in this analysis. Pts received rev (163 mg or 95 mg/m2 if body weight &amp;lt;40 kg) q12h with a strong cytochrome P450 3A4 inhibitor orally in 28-day cycles. Treatment continued until unacceptable toxicity or lack of at least morphological leukemia-free state (MLFS) after 4 cycles of treatment. Phase 2 primary objectives were safety and tolerability of rev and CR+CR with partial hematologic recovery (CRh) rate. Key secondary endpoints included composite CR rate (CRc, CR+CRh+CR with incomplete platelet recovery [CRp]+CR with incomplete count recovery [CRi]) and overall response rate (ORR, CRc+MLFS+partial remission). A planned interim analysis (IA) of pooled adult and pediatric pts with KMT2Ar acute leukemia was conducted. Results:As of July 24, 2023, 94 pts with R/R KMT2Ar acute leukemia received ≥1 dose of study drug and were included in the safety analysis (Table 1). Median age was 37.0 y (range, 1.3-75.0). Of the 94 pts, 23 (24.5%) were aged &amp;lt;18 y and 13 (13.8%) were aged ≥65 y. Seventy-eight (83%) had AML and 16 (17.0%) had ALL or MPAL. Over half of pts were female; 17.5% of pts were non-White. Pts were heavily pretreated (median 2 [range, 1-11] prior lines of therapy), with 41 pts (43.6%) having received ≥3 prior lines. Fifty-four pts (57.4%) had refractory relapse disease (unresponsive to most recent salvage treatment), and 47 pts (50%) had prior HSCT. Treatment-related adverse events (TRAEs) were reported in 81.9% of the safety population. Most common TRAEs (≥20%) were nausea (27.7%), differentiation syndrome (26.6%), and QTc prolongation (23.4%). Grade ≥3 TRAEs were observed in 51 pts (54.3%), the most common being differentiation syndrome (16.0%), febrile neutropenia (13.8%), and QTc prolongation (13.8%) (Table 2). Overall, 6.4% of pts discontinued therapy due to TRAEs; none discontinued rev due to differentiation syndrome or QTc prolongation. The pooled efficacy population for the IA (n=57) included all phase 2 pts with centrally confirmed KMT2Ar and ≥5% blasts in bone marrow at baseline who had received ≥1 dose of study drug and started treatment on or before the 38th adult AML efficacy evaluable patient. The IA was specified to occur when the 57 patients have had the opportunity to be followed for 6 months. After median follow-up of 6.1 mo, 13 pts (22.8% [95% confidence interval (CI), 12.7-35.8]) achieved CR+CRh, surpassing the predefined IA efficacy boundary for the pooled KMT2Ar population; CR+CRh rate was similar in adult and pediatric pts. Median duration of CR+CRh was 6.4 mo (95% CI, 3.4-not reached). CRc was 43.9% (95% CI, 30.7-57.6); ORR was 63.2% (95% CI, 49.3-75.6). Most pts with a CR or CRh response, and for whom measurable residual disease (MRD) status was reported, achieved MRD negativity (7/10, 70.0%); most pts with CRc and MRD reported also achieved MRD negativity (15/22, 68.2%). Fourteen of 36 responders (38.9%) proceeded to HSCT, with half resuming rev post HSCT. Conclusions: Rev demonstrated clinically meaningful results in a heavily pretreated KMT2Ar population, including high ORR and rates of MRD negativity and subsequent HSCT. At IA, this pivotal study met its primary endpoint and the KMT2Ar cohorts were stopped early for efficacy.

Komet-008: A Phase 1 Study to Determine the Safety and Tolerability of Ziftomenib Combinations for the Treatment of KMT2A-Rearranged or NPM1-Mutant Relapsed/Refractory Acute Myeloid Leukemia

Background and Significance: Relapsed or refractory (R/R) acute myeloid leukemia (AML) with nucleophosmin 1-mutations ( NPM1-m) or KMT2A-rearrangment ( KMT2A-r) remain a high unmet need. Ziftomenib is a potent and selective inhibitor of the interaction between menin and mixed-lineage leukemia (MLL) lysine[K]-specific methyltransferase 2A ( KMT2A), which drives leukemogenesis in these subtypes. Preclinical data have demonstrated ziftomenib's ability to target multiple types of menin-dependent AML clones, and that the antitumor activity of ziftomenib and other menin inhibitors in cell-derived xenograft (CDX) and patient-derived xenograft (PDX) models of KMT2A-r and NPM1-m AML is enhanced by the addition of FLT3 inhibitors such as gilteritinib and quizartinib. An ongoing Phase 1/2 study (KOMET-001) in R/R AML has demonstrated that ziftomenib monotherapy is safe and well tolerated and that the clinical activity at the recommended phase 2 dose (RP2D) is robust, as the rates of response were not affected by the presence of downstream co-mutations, such as FLT3 and IDH1 or IDH2. Given the monotherapy activity seen to date, the development of ziftomenib in combination with standard-of-care (SOC) chemotherapies or FLT3 inhibitors may provide increased clinical benefit for R/R AML patients with KMT2A-r or with NPM1-m with or without concurrent FLT3-mutations ( FLT3-m) and warrants further investigation. Study Design and Methods: KOMET-008 (NCT# 05735184) is an open-label, dose escalation and expansion study to determine the safety, tolerability, and preliminary efficacy of ziftomenib when combined with SOC regimens for the treatment of either NPM1-m (Arm A) or KMT2A-r (Arm B) R/R AML. Each genetically defined arm will evaluate ziftomenib in combination with various SOC treatments in separate and independent cohorts, as outlined below: Arm A:NPM1-m R/R AML Cohort A-1: Ziftomenib plus FLAG-IDACohort A-2: Ziftomenib plus LDACCohort A-3: Ziftomenib plus gilteritinib (for NPM1-m + FLT3-m R/R AML) Arm B: KMT2A-r R/R AML Cohort B-1: Ziftomenib plus FLAG-IDACohort B-2: Ziftomenib plus LDAC The Part 1a dose escalation uses a i3+3 design, followed by dose expansion/validation in Part 1b. Key eligibility criteria include adults (≥18 years) with documented NPM1-m (±co-occurring FLT3-m) or KMT2A-r R/R AML, ECOG ≤2, prior hematopoietic stem cell transplant (HSCT) allowed, if recovered and prior gilteritinib allowed. Patients who receive an HSCT during study participation will be allowed to receive ziftomenib maintenance therapy upon recovery. The primary objectives are to establish the safety and tolerability of ziftomenib when combined with SOC therapies (ie, dose limiting toxicities, AEs per CTCAE v5.0) and to identify the RP2D of ziftomenib for each SOC combination in each genetically defined cohort. Secondary endpoints include preliminary efficacy (including MRD-negative responses and number of patients able to receive stem cell transplant) and pharmacokinetics. Exploratory end points include biomarkers for efficacy and resistance and pharmacodynamic biomarkers potentially related to the activity of ziftomenib when combined with SOC treatments in adults with R/R AML with NPM1-m or KMT2A-r.

Influence of Bone Marrow Blast Enumeration and Co-Occurring Myelodysplasia Related Gene Mutations in NPM1-Mutated Myeloid Malignancies

Introduction Both World Health Organization (WHO 5th) and International Consensus Classification (ICC) guidelines recognize mutations in nucleophosmin 1 ( NPM1mut) as a class defining molecular event in acute myeloid leukemia (AML). WHO 5th omitted the blast percentage requirement and ICC lowered the blast threshold to 10%. NPM1mut AML without FLT3 ITD or adverse risk cytogenetics is considered favorable risk in European LeukemiaNet (ELN) 2022, however co-occurring adverse-risk myelodysplasia related mutations (MR mut) do not influence ELN 2022 risk stratification. This analysis addresses the prognostic impact of blast enumeration in NPM1mut myeloid neoplasms (MN) with &amp;lt;10% blasts and influence of co-occurring mutations in NPM1mut AML. Methods We interrogated publicly available data from adult (≥18 years) patients (pts) with chronic myeloid neoplasms included in the International Working Group for Prognosis in MDS (N= 3,323; Bernard et al. NEJM Evid 2022) and two independent AML cohorts (AMLSG [N=1540] Papaemmanuil et al. NEJM 2016; UKNCRI [N=2,113] Tazi et al. Nat. Commun. 2022). Between group differences were assessed using the Wilcoxon rank-sum test or Fisher's exact test as appropriate. Time to event endpoints were analyzed using the log-rank method. Multivariate analysis (MVA) used cox proportional hazards regression. Results NPM1 mut was identified in 17% (N=1148) of pts (AML: 30% [1109/3653], MN with &amp;lt;20% blasts: 1.2% [39/3,323]). Compared to pts with NPM1mut AML, pts with NPM1mut MN (MDS, CMML, aCML) with blast &amp;lt; 20% were older (median age 65 vs. 55 years, p &amp;lt; 0.001), had a lower median total WBC count (5 vs. 32 x10 9 /L, p &amp;lt; 0.001) and bone marrow (BM) blast percentage (8% vs. 76%, p &amp;lt; 0.001). No significant difference was observed between NPM1mut AML and MN with &amp;lt;20% blasts in median hemoglobin (9.1 vs. 9 x10 9, p: 0.44), platelets (62 vs. 66 x10 9 /L, p: 0.55), or NPM1mut variant allele frequency (VAF; 35% vs. 37%, p: 0.64). After a median follow up of 4.7 years (range: 0-12), no difference in overall survival (OS) was observed between pts with low blast (LB; &amp;lt;10%) NPM1mut MN (N=19) vs. ≥10% blasts (N=18) (median OS: 0.64 years [95% CI: 0.31-3.0] vs. 1.1 years [95% CI: 0.95-NR], p=0.32). Median leukemia free survival (LFS) was 0.81 years (95% CI: 0.67-1.5) and not significantly different between NPM1mut MN pts with &amp;lt;10 vs. ≥10% blasts (median LFS: 0.55 vs. 0.91 years, p=0.27). In pts with NPM1mut AML (N=1109), 52% (N=581) were ELN 2022 favorable-risk; 15% (N=92) had MR mut including SRSF2 (51%, N=47), STAG2 (21%, N=19), ASXL1 (9%, N=8), BCOR (9%, N=8), RUNX1 ( 8%, N=7), EZH2 (5%, N=5), SF3B1 (5%, N=5), U2AF1 (1%, N=1), and ZRSR2 (1%, N=1). Pts with MR mut were older (median age 62 vs. 53 years, p &amp;lt; 0.001), had higher BM blasts (80% vs. 72% p=0.019), and lower platelets (45 vs. 77 x10 9/L p &amp;lt; 0.001) compared to pts with NPM1mut AML without MR mut. No significant difference in NPM1mut VAF was observed (34% in both groups). After a median follow up of 3.5 years (range: 0-15), median OS was 6.2 years in pts with NPM1mut AML (95% CI: 4.2-9.7). Inferior OS was observed in ELN favorable risk NPM1mut pts with vs. without MR mut, respectively (2 vs. 8.4 years, p=0.0001). When stratified by age &amp;lt; 60 vs. ≥ 60 years, younger pts with MR mut had an increased risk of death (HR 1.7 95% CI: 1.1-2.8, p: 0.02). No OS difference was observed in older pts with or without MR mut (HR: 1.2, 95% CI: 0.80-1.68, p=0.44), however OS was significantly shorter in older pts with NPM1mut compared to younger pts (median 1.7 years vs. NR, p &amp;lt; 0.001). Patients with NPM1mut /MR mut AML had survival comparable to patients with ELN 2022 intermediate risk AML (2.0 vs. 2.1 years, p=0.35). In MVA of NPM1mutAML adjusted for MR mut, BM blast percentage, age, performance status, baseline WBC, hemoglobin, platelet count, NPM1mut VAF, and treatment setting (AMLSG or UKNCRI), the presence of an MR mut (HR 1.39 [95% CI: 1.0-1.9], p=0.04), age &amp;lt; 60 (HR 0.43 [95% CI: 0.32-0.56], p &amp;lt; 0.001), and performance status (HR 1.35 [95% CI: 1.14-1.60], p=0.0004) retained statistical significance. Conclusion Pts with low blast NPM1mut MN have outcomes similar to pts with NPM1mut AML, supporting NPM1mut as an AML defining event irrespective of BM blast enumeration. In patients with ELN favorable risk NPM1mut AML, co-occurrence of MR mut is independently associated with inferior survival, comparable to ELN intermediate risk AML. These results may inform future refinements of current consensus guidelines.

Revumenib Monotherapy in Patients with Relapsed/Refractory KMT2Ar Acute Leukemias: Efficacy and Safety Results from the Augment-101 Phase 1/2 Study

Background: Revumenib (SNDX-5613), a potent, selective small-molecule inhibitor of the menin-histone-lysine N-methyltransferase 2A (KMT2A) interaction, is being investigated in patients (pts) with relapsed/refractory (R/R) KMT2A-rearranged ( KMT2Ar) and nucleophosmin 1-mutated ( NPM1m) acute leukemias. An initial analysis of the phase 1 of AUGMENT-101 (NCT04065399) has been reported ( Nature. 2023;615:920-924). Here we report an update of &amp;gt;1 year additional experience in the phase 1 portion of the study, which has completed enrollment, with safety data from 131 treated pts and efficacy data in 80 pts with R/R KMT2Ar leukemia. Demographic data from pivotal phase 2 cohorts are included; safety and efficacy data of a preplannedanalysis will be reported at the meeting. Methods: In phase 1, pts aged ≥30 days with R/R acute leukemias were assigned to 1 of 6 dose-escalation arms designed to identify a recommended phase 2 dose (RP2D) for concomitant administration of weak, moderate, or strong cytochrome P450 3A4 inhibitor (CYP3A4i) azole antifungal or no CYP3A4i. Pts received revumenib every 12 hours (q12h) or 3 times a day at a flat dose (≥40 kg) or a body surface area-based dose (&amp;lt;40 kg). Safety was assessed in all pts who received ≥1 dose of revumenib; responses are reported for all treated pts with KMT2Ar. In pts who underwent hematopoietic stem cell transplant (HSCT), responses reported are those achieved before HSCT. The Phase 2 study was initiated following identification of an RP2D of 163 mg (95 mg/m 2 if &amp;lt;40 kg) q12h with a strong CYP3A4i in 28-day cycles until unacceptable toxicity, lack of at least morphological leukemia-free state (MLFS) by end of cycle 4, or progressive disease without clinical benefit as defined by the investigator. The phase 2 primary objectives are evaluation of safety and tolerability of revumenib and assessment of the complete remission (CR) + CR with partial hematologic recovery (CRh) rate. The safety and efficacy of revumenib at an RP2D is being explored in 3 expansion cohorts: Cohort 2A, KMT2Ar acute lymphoblastic leukemia (ALL)/mixed phenotype acute leukemia; Cohort 2B, KMT2Ar acute myeloid leukemia (AML); and Cohort 2C, NPM1m AML. A preplanned pooled analysis of adult and pediatric pts with KMT2Ar acute leukemia will be conducted when a subset of Cohorts 2A+2B pts have completed a minimum of 4 cycles or have discontinued therapy. Results: Phase 1: As of June 19, 2023, 80 pts with R/R KMT2Ar acute leukemia were treated with revumenib in the 6 dose-escalation arms (Table 1). In adults with AML (n=51), the largest KMT2Ar subgroup, CR+CRh was 28% and overall response rate (ORR) was 59%. Responses were consistent in ALL/other leukemia subtype (n=15), with 27% CR+CRh and 53% ORR. ORR (61%) was similar in pediatric pts (&amp;lt;18 years, n=18), with 22% proceeding to HSCT. Although similar percentages of adult and children proceeded to transplant, consistent with common clinical practice in pediatrics, 75% of children transplanted went on to HSCT prior to achieving a CR/CRh (2 MLFS, 1 CRp) versus 17% of adults. In the safety population (n=131), 24% of pts had a ≥Grade 3 treatment-related adverse event (TRAE), with Grade 3 QTc prolongation in 8% and differentiation syndrome in 2% (Table 1). QTc prolongation remained as the only dose-limiting toxicity; no patients experienced ventricular arrhythmias. In addition, based on updated phase 1 pharmacokinetics, clinical activity, and safety data, an RP2D of 276 mg q12h without a strong CYP3A4i was established. Phase 2: In a preliminary analysis of Cohorts 2A and 2B (June 19, 2023), 88 pts with KMT2Ar acute leukemia were enrolled and treated with revumenib at an RP2D (Table 2). Ages ranged from 1.3 to 75 years (22 aged &amp;lt;18 y; Table 2). Leukemia subtypes included ALL (15%), AML (81%), and other (3%). Pts were heavily pretreated, having received a median of 3 prior lines of therapy (range 1-12); 38% received ≥4 prior lines and 48% had prior HSCT. Conclusions: These phase 1 data continue to demonstrate deep responses to revumenib and a manageable safety profile in heavily pretreated patients with R/R KMT2Ar acute leukemia across ages and subtypes. The lack of targeted therapies approved for KMT2Ar leukemia indicate the need for further investigation of novel, potentially transformative therapies such as revumenib. Enrollment in the AUGMENT-101 trial is ongoing. Pivotal Phase 2 safety and efficacy data for the KMT2Ar population will be reported at the meeting.