NPM1-mutated Acute Myeloid Leukemia Research Articles

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.

NPM1 exon 5 mutations in acute myeloid leukemia: Implications in diagnosis and minimal residual monitoring.

NPM1 exon 5 mutations in acute myeloid leukemia: Implications in diagnosis and minimal residual monitoring.

Cytogenetic abnormalities in NPM1-mutated acute myeloid leukemia

NPM1 mut acute myeloid leukemia (AML) has been identified as a distinct entity of myeloid neoplasms according to the 2017 European LeukemiaNet (ELN) guidelines. It confers a favorable prognosis regardless of cytogenetic abnormalities. We evaluated 418 newly diagnosed AML patients to test the validity of this hypothesis. Seventy-four patients with NPM1 mut AML showed a good response to induction and a relatively favorable prognosis. Abnormal karyotypes were observed in 15 patients. Chromosomal abnormalities were significantly associated with a worse prognosis in NPM1 mut AML patients (5-year overall survival (OS): 38.9 ± 12.9%, p = .037; event-free survival (EFS): 33.3 ± 12.2%, p = .043, respectively). Four patients with abnormal karyotypes who underwent allogeneic hematopoietic stem cell transplantation (alloHSCT) during CR1 had longer survival than those who received chemotherapy only. Multivariable analysis revealed abnormal karyotypes independently predicted OS and EFS among NPM1 mut AML patients. In summary, cytogenetic abnormalities are strong prognostic indicators in NPM1 mut AML. Therefore, they should be classified accordingly, and alloHSCT should be performed on selected patients during CR1.

Tandem measurable residual disease assessment by 10-colour multiparameter flow cytometry and quantitative polymerase chain reaction in core-binding factor and NPM1-mutated acute myeloid leukaemia

Background: In acute myeloid leukaemia (AML), studies comparing multiparameter flow cytometry (MFC) and quantitative polymerase chain reaction (qPCR) measurable residual disease (MRD) assessment have shown varying concordance rates. Fewer studies have examined the concordance between MFC and qPCR MRD and its prognostic impact.

Mutant NPM1-Regulated FTO-Mediated m6A Demethylation Promotes Leukemic Cell Survival via PDGFRB/ERK Signaling Axis.

Acute myeloid leukemia (AML) with nucleophosmin 1 (NPM1) mutations exhibits distinct biological and clinical features, accounting for approximately one-third of AML. Recently, the N6-methyladenosine (m6A) RNA modification has emerged as a new epigenetic modification to contribute to tumorigenesis and development. However, there is limited knowledge on the role of m6A modifications in NPM1-mutated AML. In this study, the decreased m6A level was first detected and high expression of fat mass and obesity-associated protein (FTO) was responsible for the m6A suppression in NPM1-mutated AML. FTO upregulation was partially induced by NPM1 mutation type A (NPM1-mA) through impeding the proteasome pathway. Importantly, FTO promoted leukemic cell survival by facilitating cell cycle and inhibiting cell apoptosis. Mechanistic investigations demonstrated that FTO depended on its m6A RNA demethylase activity to activate PDGFRB/ERK signaling axis. Our findings indicate that FTO-mediated m6A demethylation plays an oncogenic role in NPM1-mutated AML and provide a new layer of epigenetic insight for future treatments of this distinctly leukemic entity.

Association of Next Generation Sequencing Based Genotypic Profiling with MICM Characteristics in NPM1 Mutated Acute Myeloid Leukemia

To explain the clinicobiological heterogeneity of NPM1 mutated (NPM1mut) acute myeloid leukemia (AML) by analyzing the association between next-generation sequencing (NGS) profiles and MICM characteristics in patients with this AML subtype. Data of 238 NPM1mut patients with available NGS information on 112 genes related to blood disease was collected, and χ2 test and nonparametric test were used to analyze the distribution association between NGS-detecting mutations and conventional MICM parameters. In entire NPM1mut cohort, totaling 240 NPM1 mutation events were identified, of whom 10 (10/240, 4.2%) were missense mutations, which did not involve any W288 or W290 locus and were found exclusively in NPM1mut/FLT3-ITD- group. All but one of these missense mutations (9/10, 90%) were accompanied by AML subtype-defining recurrent cytogenetic or molecular abnormalities, of which 7 cases were in the low risk and 2 in the high risk. NPM1mut occurred solely as an insertion/deletion (indel) type in the NPM1mut/FLT3-ITD+ group. The incidence of favorable plus unfavorable karyotypes in NPM1mut/FLT3-ITD- group was higher than in NPM1mut/FLT3-ITD+ group (6.4% vs. 0, P=0.031). The positive rates of CD34 and CD7 in NPM1mut/FLT3-ITD+ group were significantly higher than in NPM1mut/FLT3-ITD- group (CD34: 47.9% vs. 20.6%, P<0.001; CD7: 61.5% vs. 29.9%, P<0.001). Logistic analysis showed that FLT3-ITD independently predicted for CD34+ and CD7+ [odds ratio (OR)=5.29, 95%CI: 2.64-10.60, P<0.001; OR=3.47, 95%CI: 1.79-6.73, P<0.001; respectively]. Ras-pathway mutations independently predicted for HLA-DR+ (OR=4.05, 95%CI: 1.70-9.63, P=0.002), and KRAS mutation for MPO- (OR=0.18, 95%CI: 0.05-0.62, P=0.007). TET2/IDH1 mutations independently predicted for CD34- and CD7- (OR=0.26, 95%CI: 0.11-0.62, P=0.002; OR=0.30, 95%CI: 0.14-0.62, P=0.001; respectively), and MPO+ (OR=3.52, 95%CI: 1.48-8.38, P=0.004). DNMT3A-R882 independently predicted for CD7+ and HLA-DR+ (OR=3.59, 95%CI: 1.80-7.16, P<0.001; OR=13.41, 95%CI: 4.56-39.45, P<0.001; respectively), and DNMT3A mutation for MPO-(OR=0.35, 95%CI: 1.48-8.38, P=0.004). Co-existing FLT3-ITD in NPM1mut AML independently predicts for CD34+ and CD7+, co-existing Ras-pathway mutation for HLA-DR+ and MPO-, co-existing TET2/IDH1 mutation for CD34-, CD7-, and MPO+, and co-existing DNMT3A mutation for HLA-DR+, CD7+, and MPO-, thereby providing a new mechanism explanation for the immunophenotypic heterogeneity of these AML patients.

Measurable residual disease, FLT3-ITD mutation, and disease status have independent prognostic influence on outcome of allogeneic stem cell transplantation in NPM1-mutated acute myeloid leukemia.

Nucleophosmin‐1 (NPM1) mutations in acute myeloid leukemia (AML) confer a survival advantage in the absence of FLT3‐internal tandem duplication (FLT3‐ITD). Here, we investigated the main predictors of outcome after allogeneic hematopoietic stem cell transplantation (allo‐HCT). We identified 1572 adult (age ≥ 18 year) patients with NPM1‐mutated AML in first complete remission (CR1:78%) or second complete remission (CR2:22%) who were transplanted from matched sibling donors (30.8%) or unrelated donors (57.4%) between 2007 and 2019 at EBMT participating centers. Median follow‐up for survivors was 23.7 months. FLT3‐ITD was present in 69.3% of patients and 39.2% had detectable minimal/measurable residual disease (MRD) at transplant. In multivariate analysis, relapse incidence (RI) and leukemia‐free survival (LFS) were negatively affected by concomitant FLT3‐ITD mutation (HR 1.66 p = 0.0001, and HR 1.53, p < 0.0001, respectively), MRD positivity at transplant (HR 2.18, p < 10−5 and HR 1.71, p < 10−5, respectively), and transplant in CR2 (HR 1.36, p = 0.026, and HR 1.26, p = 0.033, respectively), but positively affected by Karnofsky score ≥90 (HR 0.74, p = 0.012, and HR 0.7, p = 0.0002, respectively). Overall survival (OS) was also negatively influenced by concomitant FLT3‐ITD (HR 1.6, p = 0.0001), MRD positivity at transplant (HR 1.61, p < 10−5), and older age (HR 1.22 per 10 years, p < 0.0001), but positively affected by matched sibling donor (unrelated donor: HR 1.35, p = 0.012; haploidentical donor: HR 1.45, p = 0.037) and Karnofsky score ≥90 (HR 0.73, p = 0.004). These results highlight the independent and significant role of FLT3‐ITD, MRD status, and disease status on posttransplant outcomes in patients with NPM1‐mutated AML allowing physicians to identify patients at risk of relapse who may benefit from posttransplant prophylactic interventions.

The Role of Nucleophosmin 1 (NPM1) Mutation in the Diagnosis and Management of Myeloid Neoplasms.

Nucleophosmin (NPM1) is a multifunctional protein with both proliferative and growth-suppressive roles in the cell. In humans, NPM1 is involved in tumorigenesis via chromosomal translocations, deletions, or mutation. Acute myeloid leukemia (AML) with mutated NPM1, a distinct diagnostic entity by the current WHO Classification of myeloid neoplasm, represents the most common diagnostic subtype in AML and is associated with a favorable prognosis. The persistence of NPM1 mutation in AML at relapse makes this mutation an ideal target for minimal measurable disease (MRD) detection. The clinical implication of this is far-reaching because NPM1-mutated AML is currently classified as being of standard risk, with the best treatment strategy (transplantation versus chemotherapy) yet undefined. Myeloid neoplasms with NPM1 mutations and <20% blasts are characterized by an aggressive clinical course and a rapid progression to AML. The pathological classification of these cases remains controversial. Future studies will determine whether NPM1 gene mutation may be sufficient for diagnosing NPM1-mutated AML independent of the blast count. This review aims to summarize the role of NPM1 in normal cells and in human cancer and discusses its current role in clinical management of AML and related myeloid neoplasms.

Acquired WT1 mutations contribute to relapse of NPM1-mutated acute myeloid leukemia following allogeneic hematopoietic stem cell transplant.

The role of WT1 protein in hematopoiesis and leukemogenesisis incompletely elucidated. WT1 overexpression is commonin acute myeloid leukemia (AML); however, WT1 mutations occur in only about 10% of cases, with increasing incidence in the setting of relapse. In this study, we investigated the clinical and molecular characteristics of WT1 mutations in NPM1-mutated AML, to enhance our understanding of the biology and potential therapeutic implications of WT1 mutations. Our study cohort included 67 patients with NPM1 mutated AML and a median follow-up of 13.7 months. WT1 mutations were identified in 7% (n = 5) of patients at the time of initial diagnosis. WT1 mutant clones were presumed to be present as co-dominant clones in 3/5 and in subclonal populations in 2/5 cases based on variant allelic frequency (VAF) when compared with NPM1 mutation VAF. All WT1 mutations became undetectable at time of MRD-negative (NPM1-wild type) remission. None of these patients experienced relapse at the time of last follow-up (median, 15 months; range, 4.5-20.2 months). A total of 15/67 (22%) patients relapsed; among these patient, four (27%) relapsed with WT1 mutant AML. Three of four patients had undergone allogeneic hematopoietic stem cell transplantation (HSCT). None of these patients had detectable WT1 mutations at the time of initial diagnosis. WT1 mutations were presumed clonal in two cases and subclonal in the other two cases, based on VAF. Our results indicate that WT1 mutations contribute to relapse in NPM1 mutated AML, especially in the setting ofHSCT. These findings suggest that emerging WT1 mutations may serve as a conduit for relapse in NPM1-mutated AML, and thatsequential molecular profiling to evaluate potential emergent WT1 mutations during surveillance and particularly at relapse likely has prognostic value in patients with NPM1 mutatedAML.

3055 – METALLOTHIONEIN 1 (MT1) AS A TARGETABLE DEPENDENCY IN ACUTE MYELOID LEUKEMIA

Adult de novo acute myeloid leukemia (AML) is a blood cancer with poor prognosis. The heterogeneous nature of this disease motivates identification and understanding of specific dependencies within genetic and/or epigenetic subtypes of AML. The goal of this project is to understand the molecular mechanisms underlying AML development in the context of two recurrent and frequently co-mutated genes in human AML; the DNA methyltransferase DNMT3A and nucleophosmin NPM1. We used single-cell RNA-seq and cellHarmony analysis of our recently developed mouse model replicating DNMT3A and NPM1 mutations found in human AML to identify differentially expressed transcripts. Across four independently derived Dnmt3a;Npm1-mutant AML samples, metallothionein 1 (Mt1) was the top common, significantly upregulated transcript in the most primitive AML stem/progenitor cell compartment. MT-1 functions to protect against heavy metal toxicity, inflammation, oxidative stress, and has been implicated in cancer progression. This led us to hypothesize that upregulation of MT-1 is critical for growth and survival of DNMT3A;NPM1-mutant AML. First, we validated increased Mt1 expression using additional independent Dnmt3a;Npm1-mutant AML bone marrow and spleen samples compared to control wild-type cells. Next, using the DNMT3A;NPM1-mutant human AML cell line OCI-AML3, we found that MT-1 expression was increased 100-fold relative to a control human 293T cell line. To test the extent to which loss of Mt1 can inhibit growth of Dnmt3a;Npm1-mutant AML cells, we performed CRISPR-mediated knockout followed by ex vivo culture. We found that Mt1-knockout Dnmt3a;Npm1-mutant AML cells have reduced proliferation and expansion compared to control-targeted cells. Taken together, our study nominates MT-1 as a target towards the goal of treatment and prevention of DNMT3A;NPM1-mutant AML.

Integrated Genomic Analysis Identifies UBTF Tandem Duplications As a Subtype-Defining Lesion in Pediatric Acute Myeloid Leukemia

Children with acute myeloid leukemia (AML) have a dismal prognosis due to a high relapse rate; however, the molecular basis leading to relapsed pediatric AML has not yet been fully characterized. To define the spectrum of alterations common at relapse, we performed integrated profiling of 136 relapsed pediatric AML cases with RNA sequencing (RNA-seq), whole-genome sequencing, and target-capture sequencing. In addition to well-characterized fusion oncoproteins, such as those involving KMT2A (n=36, 26.5%) or NUP98 (n=18, 13.2%), we also identified somatic mutations in UBTF (upstream binding transcription factor) in 12 of 136 cases (8.8%) of this relapsed cohort.Somatic alterations of the UBTF gene, which encodes a nucleolar protein that is a component of the RNA Pol I pre-initiation complex to ribosomal DNA promoters, have rarely been observed in AML. In our cohort, all alterations can be described as heterozygous in-frame exon 13 tandem duplications (UBTF-TD), either at the 3' end of exon 13 of UBTF or of the entire exon 13 (Fig. A). As we noticed limited detection in our pipeline as a result of complex secondary indels alongside the duplications, we established a soft-clipped read-based screening method to detect UBTF-TD more efficiently. Applying the screening to RNA-seq data of 417 additional pediatric AMLs from previous studies and our clinical service, we identified 15 additional UBTF-TDs, many of which have not been previously reported. At the amino acid level, UBTF-TDs caused amino acid insertions of variable sizes (15-181 amino acids), duplicating a portion of high mobility group domain 4 (HMG4), which includes short leucine-rich sequences.UBTF-TD AMLs commonly occurred in early adolescence (median age: 12.6, range: 2.4-19.6), and 19 of the total 27 cases had either normal karyotype (n=12) or trisomy 8 (n=7). UBTF-TD is mutually exclusive from other recurrent fusion oncoproteins, such as NUP98 and KMT2A rearrangements (Fig. B), but frequently occurred with FLT3-ITD (44.4%) or WT1 mutations (40.7%). The median variant allele fraction (VAF) of the UBTF-TD was 48.0% (range: 9.7-66.7%). In four cases with data at multiple disease time points, the identical UBTF-TDs were present at high allele fractions at all time points, suggesting that UBTF-TD is a clonal alteration. tSNE analysis of the transcriptome dataset showed that UBTF-TD AMLs share a similar expression pattern with NPM1 mutant and NUP98-NSD1 AML subtypes, including NKX2-3 and HOXB cluster genes (Fig. C) . Altogether, these findings suggest that UBTF-TD is a unique subtype of pediatric AML.To address the impact of UBTF-TD expression in primary hematopoietic cells, we introduced UBTF-TD and UBTF wildtype expression vectors into cord blood CD34+ cells via lentiviral transduction. UBTF-TD expression promotes colony-forming activity and cell growth, yielding cells with a persistent blast-like morphology (Fig. D). Further, transcriptional profiling of these cells demonstrated expression of HOXB genes and NKX2-3, similar to UBTF-TD AMLs in patients, indicating that UBTF-TD is sufficient to induce the leukemic phenotype.To investigate the prevalence of UBTF-TDs in larger de novo AML cohorts, we applied the above UBTF-TD screening method to the available de novo AML cohorts of TCGA (n=151, adult), BeatAML (n=220, pediatric and adult), and AAML1031 (n=1035, pediatric). We identified UBTF-TDs in 4.3% (45/1035) of the pediatric AAML1031 cohort, while the alteration is less common (0.9%: 3/329, p=0.002) in the adult AML cohorts (Fig. E). In the AAML1031 cohort, UBTF-TDs remain mutually exclusive with known molecular subtypes of AML and commonly occur with FLT3-ITD (66.7%) and WT1 (40.0%) mutations and either normal karyotype or trisomy 8. The presence of UBTF-TDs in the AAML1031 cohort is associated with a poor outcome (Fig. F, median overall survival, 2.3 years) and MRD positivity; multivariate analysis revealed that UBTF-TD and WT1 are independent risk factors for overall survival within FLT3-ITD+ AMLs.In conclusion, we demonstrate UBTF-TD defines a unique subtype of AMLs that previously lacked a clear oncogenic driver. While independent of subtype-defining oncogenic fusions, UBTF-TD AMLs are associated with FLT3-ITD and WT1 mutations, adolescent age, and poor outcomes. These alterations have been under-recognized by standard bioinformatic approaches yet will be critical for future risk-stratification of pediatric AML. [Display omitted] DisclosuresIacobucci: Amgen: Honoraria; Mission Bio: Honoraria. Miller: Johnson & Johnson's Janssen: Current Employment. Mullighan: Pfizer: Research Funding; Illumina: Membership on an entity's Board of Directors or advisory committees; AbbVie: Research Funding; Amgen: Current equity holder in publicly-traded company.

Abstract P195: Omacetaxine mepessucinate plus venetoclax show strong synergistic anti-leukemic activity in nucleophosmin (NPM1)-mutated AML patient-derived xenograft (PDX) models to support a phase 1 clinical trial

Abstract Despite different novel targeted drugs against specific genetic lesions have been developed for acute myeloid leukemia (AML) treatment, therapy in many AML subtypes should still rely on drugs possibly targetting alternative pathways essential for leukemia survival. Different biological and clinical evidences point to nucleophosmin (NPM1) gene mutations (the most frequent genetic lesion in AML) among strong predictors of sensitivity to venetoclax, a small molecule drug inhibitor of the B-cell leukemia/lymphoma-2 (Bcl-2) anti-apoptotic protein, highly expressed in most AML. Venetoclax appeared to be extremely efficacious in combination with hypomethylating agents (HMA) as first-line therapy, particularly in NPM1-mutated AML. Omacetaxine mepessucinate is a synthetic form of the plant cephalotaxine alkaloid homoharringtonine (HHT). HHT binds to the A-site cleft of ribosomes preventing the initial elongation step of protein synthesis and leading to a transient but profound inhibition of protein synthesis especially of those with a short half-life, including the antiapoptotic protein myeloid cell leukemia-1 Mcl-1, acting as mechanism of resistance following Bcl-2 inhibition. Besides its action as ‘Mcl-1 inhibitor’ (targeting its protein levels), we focused on HHT because i) it is a FDA-approved drug, in the setting of chronic myeloid leukemia (CML); ii) it showed some activity in clinical trials in AML; iii) it was shown to be a safe treatment, when evaluated also in association with chemotherapy, either standard intensive or low-dose regimen, in AML; iv) it is administered subcutaneously in its FDA-approved formulation; v) it has never been used in combination with venetoclax. Here, we sought to explore the therapeutic potential of omacetaxine mepessucinate and venetoclax in pre-clinical in vivo models, and used 2 different NPM1-mutated AML PDX (one, NPM1/FLT3-ITD double mutant; the other, DNMT3A/NPM1/FLT3-ITD triple mutant) edited to express luciferase allowing to track the disease by in vivo bioluminescence imaging (BLI) during treatment and evaluate drug response. HHT was given at 1 mg/Kg by subcutaneous administration for 2 consecutive weeks (5 days/week) and venetoclax at 100 mg/kg by gavage for 4 weeks (5 days/week), in cycles of 28 days. The anti-leukemic activity of the treatment was measured by evaluating AML growth/tissue infiltration on sacrificed animals by immunohistochemistry or flow cytometry and in vivo by BLI. Strikingly, on overt disease combinatorial HHT and venetoclax treatment resulted in a strong and synergic anti-leukemic effect with no leukemia detection in a cohort of mice sacrificed and analyzed after the first cycle, and in a significant advantage in a cohort analyzed for survival compared with single-drug or vehicle-treated animals. These data gave the support to design a pilot phase 1 clinical trial aimed at assessing safety and preliminary efficacy of omacetaxine mepessucinate plus venetoclax in relapsed/refractory NPM1-mutated AML patients. The trial is now open to patients accrual at our Hematology department. Citation Format: Federica Mezzasoma, Valeria Cardinali, Ilaria Gionfriddo, Francesca Milano, Sofia Sciabolacci, Alessio Ferrari, Marcella Sabino, Serenella Silvestri, Serena Donini, Valentina Tini, Giulio Spinozzi, Brunangelo Falini, Maria Paola Martelli. Omacetaxine mepessucinate plus venetoclax show strong synergistic anti-leukemic activity in nucleophosmin (NPM1)-mutated AML patient-derived xenograft (PDX) models to support a phase 1 clinical trial [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P195.

The Mutated Gene Sequenced by NGS and the Correlation of Their Coexistent Mutual Exclusiveness in NPM1 Mutated Acute Myeloid Leukemia

To analyze the clinicobiological heterogeneity of NPM1 mutated (NPM1mut) acute myeloid leukemia (AML) detected by next generation sequencing (NGS) and their coexistence and mutual exclusivity relationship in the AML subtype. The NGS data based on 112 genes related to blood disease in 238 newly diagnosed patients with NPM1mut were collected. The χ2 test and non-parametric test were used to analyze the distribution correlation between the genes in the mutational spectrum. Among all the patients, at least one co-mutation was detected out. The median number per case of the mutated genes, including NPM1mut was 4.5 (range 2－14), among them, there were 5.0 (range 2－10) for NPM1mut/FLT3-ITD+ and 4.0 (range 2－14) for NPM1mut/FLT3-ITD- cases, but it was no significant difference between the two groups (P=0.378). A total of 240 NPM1 mutational events were detected out in entire 238 NPM1mut patients, of which 10 (4.2%) were missense mutations, and were all found in NPM1mut/FLT3-ITD- patients. Most (9/10, 90%) of these NPM1 missense mutations were accompanied by AML subtype-defining cytogenetic or molecular abnormalities, of which 7 patients were in low risk or 2 in high risk. The most common NPM1mut coexisting mutations were DNMT3A (104, 43.7%), followed were FLT3-ITD (95, 39.9%) and FAT1 (57, 23.9%), FLT3-ITD and DNMT3A showed significant coexistence (P=0.005). FLT3-ITD showed significantly reciprocal exclusivity with FLT3-nonITD (P<0.001), NRAS (P<0.001), PTPN11 (P=0.017) and IDH1 (P=0.005), and showed an exclusivity inclination with KRAS (P=0.073). In addition, FLT3-nonITD along with KRAS (P=0.035), NRAS along with KRAS (P=0.008) and PTPN11 (P=0.039) coexisted significantly. Prognoses of AML involving less common NPM1 missense mutations should be stated on a case by case basis. The mutational landscape and co-occurrence and mutual exclusivity correlations of NPM1mut AML provide a mechanism explaining biological diversity and clinical heterogeneity in this AML subset.

Biological Effects of BET Inhibition by OTX015 (MK-8628) and JQ1 in NPM1-Mutated (NPM1c) Acute Myeloid Leukemia (AML)

BET inhibitors (BETi) including OTX015 (MK-8628) and JQ1 demonstrated antileukemic activity including NPM1c AML cells. Nevertheless, the biological consequences of BETi in NPM1c AML were not fully investigated. Even if of better prognosis AML patients with NPM1c may relapse and treatment remains difficult. Differentiation-based therapy by all trans retinoic acid (ATRA) combined with arsenic trioxide (ATO) demonstrated activity in NPM1c AML. We found that BETi, similar to ATO + ATRA, induced differentiation and apoptosis which was TP53 independent in the NPM1c cell line OCI-AML3 and primary cells. Furthermore, BETi induced proteasome-dependent degradation of NPM1c. BETi degraded NPM1c in the cytosol while BRD4 is degraded in the nucleus which suggests that restoration of the NPM1/BRD4 equilibrium in the nucleus of NPM1c cells is essential for the efficacy of BETi. While ATO + ATRA had significant biological activity in NPM1c IMS-M2 cell line, those cells were resistant to BETi. Gene profiling revealed that IMS-M2 cells probably resist to BETi by upregulation of LSC pathways independently of the downregulation of a core BET-responsive transcriptional program. ATO + ATRA downregulated a NPM1c specific HOX gene signature while anti-leukemic effects of BETi appear HOX gene independent. Our preclinical results encourage clinical testing of BETi in NPM1c AML patients.

Novel NPM1 exon 5 mutations and gene fusions leading to aberrant cytoplasmic nucleophosmin in AML

AbstractNucleophosmin (NPM1) mutations in acute myeloid leukemia (AML) affect exon 12, but also sporadically affect exons 9 and 11, causing changes at the protein C-terminal end (tryptophan loss, nuclear export signal [NES] motif creation) that lead to aberrant cytoplasmic NPM1 (NPM1c+), detectable by immunohistochemistry. Combining immunohistochemistry and molecular analyses in 929 patients with AML, we found non–exon 12 NPM1 mutations in 5 (1.3%) of 387 NPM1c+ cases. Besides mutations in exons 9 (n = 1) and 11 (n = 1), novel exon 5 mutations were discovered (n = 3). Another exon 5 mutation was identified in an additional 141 patients with AML selected for wild-type NPM1 exon 12. Three NPM1 rearrangements (NPM1/RPP30, NPM1/SETBP1, NPM1/CCDC28A) were detected and characterized among 13 979 AML samples screened by cytogenetic/fluorescence in situ hybridization and RNA sequencing. Functional studies demonstrated that in AML cases, new NPM1 proteins harbored an efficient extra NES, either newly created or already present in the fusion partner, ensuring its cytoplasmic accumulation. Our findings support NPM1 cytoplasmic relocation as critical for leukemogenesis and reinforce the role of immunohistochemistry in predicting AML-associated NPM1 genetic lesions. This study highlights the need to develop new assays for molecular diagnosis and monitoring of NPM1-mutated AML.

NPM1 Mutations Do Not Retain a Favorable Prognostic Impact in Adults with Advanced Relapsed or Refractory (R/R) Acute Myeloid Leukemia (AML)

NPM1 Mutations Do Not Retain a Favorable Prognostic Impact in Adults with Advanced Relapsed or Refractory (R/R) Acute Myeloid Leukemia (AML)

Mutations Highly Specific for Secondary AML Are Associated with Poor Outcomes in Patients with NPM1-Mutated ELN Favorable Risk AML

Background: NPM1 is commonly mutated in acute myeloid leukemia (AML) and represents a distinct entity under the WHO 2016 classification. It is one of the few mutations that can potentially support favorable risk by European LeukemiaNet (ELN) 2017 criteria. Mutations that are highly specific for secondary AML including SRSF2, SF3B1, U2AF1, ZRSR2, ASXL1, EZH2, BCOR, and STAG2 (sMut) (Lindsley et al.) have been shown to confer poor prognosis. The impact of these mutations on NPM1-mutated AML warrants further investigation.Objective: In this study, we explore the outcomes in patients with NPM1-mutated AML.Methods: This was a retrospective study of NPM1-mutated AML patients who were diagnosed and treated at the Moffitt Cancer Center from 2013 to March 2021. Inclusion was restricted to NPM1-mutated patients with mutation analysis (NGS) performed at diagnosis (n=159). Kaplan-Meier, univariate, and multivariate analyses were performed.Results: Among 159 patients (78M/81F, median age 63 years at diagnosis), 80.5% had de novo AML. By ELN 2017 criteria, 63.5% (101/159) had favorable risk, 27.7% (44/159) had intermediate risk, and 8.2% (13/159) had adverse risk disease. Almost 90% had intermediate risk cytogenetics at the time of diagnosis. Common co-mutations included DNMT3A (47.2%), FLT3-ITD (35.8%), TET2 (26.4%), IDH1 (17.6%), FLT3-TKD (15.1%), and IDH2 (13.8%). sMut comprised 19.5% (31/159) of patients and 20.8% (21/101) of those with ELN favorable risk. In patients with treatment response data, those with sMut never achieved CR/CRi in 35.7% (10/28) compared to 17.2% (22/128) of patients without sMut (p=0.038). The overall survival (OS) was 43.7 months with a median follow up of 35.5 months. Patients with sMut had worse OS compared to those without sMut (14.7 months vs 57.6 months, p=0.011). Among patients with favorable risk disease, OS was 11.6 months compared to not reached for those with sMut and without sMut, respectively (p<0.0001). Univariate analysis showed sMut and allogeneic hematopoietic cell transplant (HCT) significantly impacted OS (sMut: HR 3.48, 95% CI: 1.80-6.72, p<0.001; HCT: HR 0.17, 95% CI: 0.07-0.44, p<0.001). Multivariate regression using covariates including age, AML type, sMut, and HCT confirmed their prognostic significance on survival (sMut: HR 2.40, 95% CI: 1.17-4.93, p=0.017; HCT: HR 0.26, 95% CI: 0.08-0.56, p=0.002).Conclusions: Our findings suggest NPM1-mutated AML patients with sMut have significantly worse prognosis despite being classified primarily as favorable risk by ELN 2017 at diagnosis. This may have treatment implications altering the need for and/or timing of HCT. These findings should be assessed prospectively and validated in independent datasets. [Display omitted] DisclosuresHussaini: Adaptive: Consultancy, Honoraria, Speakers Bureau; Stemline: Consultancy; Amgen: Consultancy; Seattle Genetics: Consultancy; Celegene: Consultancy; Decibio: Consultancy; Guidepoint: Consultancy; Bluprint Medicine: Consultancy. Talati: AbbVie: Honoraria; Pfizer: Honoraria; Astellas: Speakers Bureau; BMS: Honoraria; Jazz: Speakers Bureau. Kuykendall: Incyte: Consultancy; Novartis: Honoraria, Speakers Bureau; Protagonist: Consultancy, Research Funding; Celgene/BMS: Honoraria; Abbvie: Honoraria; Blueprint: Honoraria; Pharmaessentia: Honoraria. Padron: Blueprint: Honoraria; Incyte: Research Funding; Kura: Research Funding; Stemline: Honoraria; Taiho: Honoraria; BMS: Research Funding. Sallman: Shattuck Labs: Membership on an entity's Board of Directors or advisory committees; Syndax: Membership on an entity's Board of Directors or advisory committees; Magenta: Consultancy; Takeda: Consultancy; Kite: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte: Speakers Bureau; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Intellia: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees; Aprea: Membership on an entity's Board of Directors or advisory committees, Research Funding. Sweet: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Bristol Meyers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; AROG: Membership on an entity's Board of Directors or advisory committees. Komrokji: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Geron: Consultancy; BMSCelgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Acceleron: Consultancy; AbbVie: Consultancy; Jazz: Consultancy, Speakers Bureau; Taiho Oncology: Membership on an entity's Board of Directors or advisory committees; PharmaEssentia: Membership on an entity's Board of Directors or advisory committees. Lancet: AbbVie: Consultancy; Celgene/BMS: Consultancy; Daiichi Sankyo: Consultancy; ElevateBio Management: Consultancy; Millenium Pharma/Takeda: Consultancy; BerGenBio: Consultancy; Jazz: Consultancy; Agios: Consultancy; Astellas: Consultancy.

Prognostic Value of Measurable Residual Disease Assessed By Multiparameter Flowcytometry in Patients with NPM1-Mutated Acute Myeloid Leukemia

Prognostic Value of Measurable Residual Disease Assessed By Multiparameter Flowcytometry in Patients with NPM1-Mutated Acute Myeloid Leukemia

Longitudinal Next Generation Sequencing Reveals the Clonal Hierarchy of IDH Mutated Clones and Impact on Survival in NPM1 Mutated AML

Longitudinal Next Generation Sequencing Reveals the Clonal Hierarchy of IDH Mutated Clones and Impact on Survival in NPM1 Mutated AML

A Novel Approach to Induce ATRA Mediated Differentiation in NPM1 Mutant Acute Myeloid Leukemia

▪Introduction: Retinoic acid (RA), an active metabolite of vitamin A, activates the retinoic acid receptors (RARs), ligand-controlled transcription factors that regulate expression of target genes affecting homeostasis and cellular differentiation. The success of RA in the form of all-trans retinoic acid (ATRA) to treat acute promyelocytic leukemia (APL) has stimulated considerable interest in the development of small molecules to modulate RARs in AML cells. Besides RARs, RA can also activate the pro-survival peroxisome proliferator-activated receptor β/δ (PPARβ/δ), a member of the nuclear hormone receptor superfamily of transcription factors that regulate several metabolic pathways in a tissue-selective manner. In the aqueous intracellular milieu, RA is transported by two alternative carriers, cellular retinoid-binding protein CRABP2, or fatty-acid-binding protein 5 (FABP5). These carriers preferentially deliver RA to RARs or PPARβ/δ respectively. In cells, expressing high CRABP2 and low FABP5, RA is delivered to and activates RARs, whereas in the presence of the reverse ratio, RA is delivered to and activates PPARβ/δ. Here we demonstrate that these two different modes of RA delivery leads to opposite cellular outcomes: CRABP2-mediated RA delivery to RAR prompts differentiation and growth arrest, while in cells containing high levels of FABP5, RA delivery to PPARβ/δ promotes survival and exponential proliferation. Inhibiting FABP5 carrier-function could be a useful method, therefore, to extend RA therapeutic activity to non-APL acute myeloid leukemia (AML).Methods: Genetic as well as pharmacologic in vitro and in vivo models of AML were used to validate FABP5 as a target for therapy to, increase RA stimulation of RARs instead of PPARβ/δ. Knockdown and overexpression of key regulators of these pathways were rationally used to understand pro-survival effects and proliferative mechanism of malignant myeloid cells. The efficacy of small molecule pharmacophore targeting FABP5 was evaluated in vitro in cell free and cell culture model as well as in vivo small animal model systems as a proof of therapeutic concept.Results: In public databases of AML gene expression, the majority of non-APL AMLs demonstrated upregulation of FABP5 and downregulation of CRABP2, potentially explaining limited benefit of RA to treat non-APL AMLs. On the other hand, ATRA sensitive APL samples demonstrated low FABP5, as expected from the established efficacy of RA to treat APLs. The greatest elevations of pro-survival FABP5 was in AMLs with mutated-NPM1. Consistent with the elevated FABP5 in these cells, we confirmed that RA activates pro-survival PPARβ/δ rather than pro-differentiation RAR in NPM1-mutated AML cells. We thus evaluated if inhibiting FABP5 could produce ATRA sensitization in NPM1 mutant AML cells. Genetic ablation of FABP5 using shRNA sensitized AML cells to ATRA treatment, while this effect was not seen in AML cells transfected with non-targeting scrambled shRNA. Cells stably expressing shRNA targeting FABP5 has significant growth perturbation in vivo in murine AML model. To target FABP5 using a small molecule approach, we used in silico drug-design to develop a novel small molecule FABP5 inhibitor (iFABP5): the inhibitor bound to recombinant FABP5 (Kd= 2x10 -7) with a 56-fold higher affinity than ATRA (Kd=11x10 -6 Mole), demonstrated in a cell free fluorescence reporter binding assay using 8-Anilino-1-naphthalenesulfonic acid (ANS). We then treated RA-resistant AML cells with iFABP5 alone and in combination with RA: neither iFABP5 nor RA alone had anti-proliferative activity as expected, but combination iFABP5 + RA downregulated PPARβ/δ target genes and upregulated RAR target genes, terminating AML cells growth.Conclusion: The lack of effective ATRA response in AML might be, in part, due to high expression of FABP5 and delivery of RA to pro-survival PPARβ/δ. A small molecule inhibitor of FABP5 could redirect RA to pro-terminal-differentiation RARs, and could be an approach to extending the remarkable activity of RA in APL to other AMLs. Reprogramming of the RA delivery to RAR pathway with the addition of a small molecular inhibitor of FABP5 could potentially restore therapeutic effects of ATRA in FABP5 over expressing cases of AML subtypes. DisclosuresMaciejewski: Regeneron: Consultancy; Novartis: Consultancy; Bristol Myers Squibb/Celgene: Consultancy; Alexion: Consultancy. Saunthararajah: EpiDestiny: Consultancy, Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Carraway: Agios: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Other: Independent review committee; Celgene, a Bristol Myers Squibb company: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Stemline: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Other: Independent review committee; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astex: Other: Independent review committee.