NPM1 Mutations Research Articles

Hyperleukocytosis in Patients with Acute Myeloid Leukemia Characteristics and Outcome, a Single-Center Experience

Background Hyperleukocytosis is the condition where Acute myeloid leukemia (AML) is presented with white blood cell counts equal to or greater than 100,000. A small proportion of AML patients are presented with this condition which is associated with significant morbidity and mortality caused by related serious complications including leukostasis, tumor lysis syndrome (TLS) and disseminated intravascular coagulation. Hyperleukocytosis is a hematological emergency requiring immediate intervention. In this retrospective study we describe the general characteristics, cytogenetics, mutational profile, treatment given, and outcome of AML patients having hyperleukocytosis at diagnosis. Methods Data were retrieved from the clinical data base of our tertiary cancer center (National Center for Cancer Care and Research, Doha, Qatar). All patients with AML diagnosed during the period January 2017 through December 2021 were included. Results A total of 186 AML patients were included. Twenty-one patients (11.5%) were presented with hyperleukocytosis. Males were more predominant 13/21 (62 %) and more than half of the patients had an Asian origin 52%. The median age for the group was 40.0 years (18-89). Complete blood count (CBC) showed median hemoglobin of 9.2g/dl, white blood cells of 176x10^3/uL and platelets 6x10^3/uL. the median percentage of the blast in the peripheral blood was 71 % and 72 % in bone marrow. Bone marrow cellularity was high in all the cases 100%. Bone marrow dysplasia was assessed with dysgranulopoiesis observed in 24%. All patients had de novo AML. In these patients the diploid KT was most common cytogenetic findings (57%), followed by CBF recurrent cytogenetics abnormalities in 4 patients (19%). FLT 3-ITD was positive in 7 patients (33%) and NPM1 mutation in 5 patients (28%). Hydroxyurea (HU) was used as cytoreductive therapy in 90% of patients. More than half of the patients 11/21 (52%) were admitted to intensive care unit (ICU). One patient had CNS relapse. Seven patients (33%) underwent allogenic bone marrow transplant. Progression occurred in 6 patients 28 (median1.4 years) and Overall survival in this cohort was 76 % (median1.9 year) and the average duration of follow-up of 24.8 months. Early death within 30 days observed, with median time to death 4 days (1-12) among those who died. Conclusion One of ten AML patients are presented with hyperleukocytosis. Most of our AML patients with hyperleukocytosis were young males. This is most probably related to the young general population in our country. All patients had hypercellular marrow with high blast percentage. Diploid Karyotype, CBF rearrangements, FLT 3-ITD mutation and NPM1 mutation were frequent in this cohort. Most of the patients developed complications requiring intensive care admission. Hydroxyurea was the first choice for cytoreduction. Both disease progression and mortality were relatively high (28% and 24 % respectively) and occurred very early, within less than a week from presentation. Early diagnosis and immediate management are mandatory to tackle the complications of this hematological emergency and improve morbidity and mortality.

Acute Myeloid Leukemia Patients Who Stopped Venetoclax or/and Azacytidine for Other Reasons Than Progression Have a Prolonged Treatment Free Remission and Overall Survival. a Filo Study

Background Recent update of the VIALE-A study showed a long-term survival for approximately 30% of acute myeloid leukemia (AML) patients treated with venetoclax and azacitidine (VEN-AZA) for a newly-diagnosed AML (ND-AML) (Pratz K.W et al., ASH 2022). A recent report of a small cohort of responding patients who interrupted VEN-AZA displayed a sustained treatment-free remission (TFR) period suggesting that treatment interruptions might be considered (Chua CC. et al., Blood Adv. 2022). To evaluate the impact of VEN-AZA discontinuation on survival and time without treatment, we retrospectively analyzed the characteristics and outcomes of patients who stopped VEN and/or AZA for reasons other than progression (e.g. hematological or other toxicities). We presented the first 51 patients from our cohort last year at ASH meeting (Garciaz S. et al, ASH 2022). In this abstract, we analyzed separately ND-AML and relapsed/refractory (R/R) AML. Methods Eligible patients 1) have been treated with VEN-AZA for a ND or R/R AML, 2) have stopped AZA and/or VEN for more than 3 months, 3) are in remission (CR, CRi or Morphologic leukemia-free state, [MLFS]) at the time of treatment interruption, 4) are ≥ 18-year-old. Main endpoints were overall survival (OS) and treatment-free remission (TFR) calculated from the time between the last day of VEN to relapse, retreatment, or death. We measured the impact of prognostic factors on OS by a Cox regression taking disease status (ND vs R/R), the discontinued treatment (VEN vs VEN-AZA), the cytogenetics and NPM1 or IDH mutations. Results We included 83 patients from French Innovative Leukemia Organization (FILO) centers and Moffit Cancer Center (US) treated with VEN-AZA for a ND-AML or a R/R AML between 11/18 and 07/23. Regarding the ND-AML patients (n=60; 32 males and 28 females), the median age was 76 (range, 26-86.5), median platelet count, WBC, ANC and bone marrow blast involvement was 52 G/L (range, 9-296), 2.8 G/L (range, 0.6-200), 0.6 G/L (range, 0.02-31.6) and 40% (range, 10-99), respectively. Cytogenetics was intermediate (int.) in 48 cases (80%) and unfavorable (unfav.) in 12 patients (20%). Eleven AML (18.3%) had NPM1 mutations, and 19 (31.6%), IDH alterations including 7 IDH1, 8 IDH2R140 and 4 IDH2R172. Six (10%) and 5 (8.3%) patients had FLT3 or TP53 mutations, respectively. The median number of VEN-AZA cycle was 4 (1-17). The best response was CR (n=44, 73.3%), CRi (n=13, 18.3%) and MLFS (n=3, 5%). Twenty-one patients out of 25 (78%) reached MRD negativity assessed by molecular (n=11) or flow cytometry (n=10) techniques. Twenty-six patients (43.3%) stopped VEN-AZA and 34 (56.7%) stopped VEN and continued AZA monotherapy for a median number of 7 AZA cycles (1-41). Reasons for treatment discontinuation were prolonged cytopenia, non-hematological toxicities, patient choice or unknown in 35 (58%), 6 (10%), 8 (13.3%), and 11 (18.3%) patients, respectively. We registered 27 relapses and 24 deaths. Fourteen patients were retreated including 11 patients who were rechallenged with VEN-AZA. Second CR/CRi rate with VEN-AZA was 3/11 (27.7%). With a median follow of 23 months, median TFR was 15 months (ranges, 3-47) and median OS was 44 months (ranges, 7-50). We also analyzed a smaller cohort of 23 R/R responding patients who stopped AZA and/or VEN. The median age was 73, median number of previous lines was 1 (1-3). Twenty-one patients (91.3%) received prior intensive chemotherapy and 4 (17.4%) prior allogeneic hematopoietic transplantation. Cytogenetic risk was int. in 17 (73.9%) and unfav. in 5 patients (21.7%) and missing in one patient. NPM1 and IDH genes were mutated in 6 (26%) and 10 (43.4%) cases, respectively. Sixteen patients (69.6%) stopped VEN-AZA and 7 (30.4%) interrupted VEN only. Nine patients resumed a treatment including 6 patients rechallenged with VEN-AZA. Among them, 3/6 (50%) obtained a second CR/CRi. Median TFR and OS were 10 (ranges, 3-23) and 19 (ranges, 7-32), respectively. Factor impacting OS in multivariate analysis were R/R AML disease status (Hazard Ratio [HR] =0.47, ranges, 0.23-0.87), and IDH mutations (HR= 2.1, ranges, 1.05-4.34), but not unfav. cytogenetics, NPM1 mutations, or the discontinued treatment (VEN vs VEN-AZA). Conclusion: Discontinuation of VEN and/or AZA in responding patients was associated with sustained responses and long-term survival rates. Resuming treatment may induce a second remission in some patients.

Prognostic Relevance of Truncating ASXL1 Mutations and ASXL1-Integrated Molecular Models in Proliferative CMML

Introduction Chronic myelomonocytic leukemia (CMML) can be classified into dysplastic (dCMML) and proliferative (pCMML) subtypes based on a white blood cell count (WBC) ≥ 13 x 10 9/L for pCMML. Though this cutoff may seem arbitrary, there is evidence that pCMML is a unique biological entity with a distinct molecular profile and worse outcomes (Carr RM, et al. Nat Commun. 2021). We hypothesized that contemporary CMML prognostic scoring systems - such as the Mayo Molecular Model (MMM; Patnaik MM, et al. Leukemia. 2014) and the CPSS-Molecular Model (CMM; Elena C, et al. Blood. 2016) - would not adequately risk-stratify pCMML patients. Moreover, while truncating ASXL1 mutations are considered a high-risk feature in CMML, their prognostic relevance in pCMML is unknown. Methods After IRB approval, we compiled a large (n = 888), molecularly-annotated database of CMML patients seen at two US medical centers (Mayo Clinic and MD Anderson) via retrospective chart review. All statistical analyses considered the clinical and laboratory parameters obtained at the time of presentation. The cohort was divided into dCMML and pCMML subgroups, which were analyzed independently alongside the entire cohort. Categorical variables were compared by Fisher exact or Pearson χ 2 tests and continuous variables by Mann-Whitney U tests. Univariate and multivariate analyses were performed using Cox proportional hazards regression models. Survival was assessed via the Kaplan-Meier method. P-values < 0.05 were considered significant. Calculations were performed using the BlueSky Statistics (v10.3.1) interface for R. Results We identified 474 (54%) dCMML cases and 410 (46%) pCMML cases with median age 71 years and 67% males. Patients with pCMML were more likely to have higher AMC (5.1 vs 1.5 x 10 9/L, p < 0.0001), circulating immature myeloid cells (IMC; 65% vs 36%, p < 0.0001) and abnormal karyotypes (37% vs 30%, p = 0.0187). They were also overrepresented in higher-risk categories of the MMM and CMM compared to dCMML (p < 0.0001 for each model). The number of somatic mutations was higher in pCMML than dCMML (mean 3.0 vs 2.6, p = 0.0050). Mutations in ASXL1 (53% vs 39%), NRAS (21% vs 11%), CBL (18% vs 12%), SETBP1 (14% vs 7%), JAK2 (10% vs 3%), CEBPA (5% vs 2%), GATA2 (3% vs 0%), and FLT3 (3% vs 1%) were more frequent while mutations in TET2 (38% vs 48%), SF3B1 (2% vs 8%), ZRSR2 (2% vs 8%), and NPM1 (1% vs 3%) were less frequent in pCMML compared to dCMML, respectively (p < 0.05 for all). Though the MMM and CMM effectively stratified the dCMML group, they did not provide meaningful risk stratification in the pCMML group (MMM p < 0.05 overall but could not distinguish between low and intermediate-1/2 or intermediate-1 and high risks; CMM p > 0.05). Likewise, there was no difference in the median overall survival (OS) or leukemia free survival (LFS) when pCMML patients were stratified by truncating ASXL1 mutations( Figure 1A). We then sought to better risk stratify pCMML. Univariate analysis identified increasing age, male gender, abnormal karyotype, and elevated WBC, ANC, and AMC as adverse predictors for OS. In addition, the presence of IMC and peripheral blood (PB) blast % were adverse factors for LFS, while age was not significant for LFS. None of the aforementioned mutations were significant in the univariate model for OS, whereas GATA2 and NPM1 mutations were significant for LFS. In multivariate analysis for OS, only male gender and AMC ≥ 10 x 10 9/L retained significance. In multivariate analysis for LFS, male gender, AMC ≥ 10 x 10 9/L, and peripheral blasts ≥ 5% remained significant; GATA2 and NPM1 mutations were not significant. These variables risk-stratified pCMML patients based on the presence of 0, 1, or ≥ 2 risk factors ( Figure 1B). In comparison, OS was 40.7 months (95% CI 35.2 - 50.1 mo.) for dCMML patients, 20.1 months (95% CI 18.2 - 22.7 mo.) for pCMML patients, and 31.8 months (95% CI 28.4 - 33.5 mo.) for the entire cohort. At last follow up, 586 deaths (66%) and 168 AML transformations (19%) were recorded in the cohort. Deaths in dCMML vs pCMML were 294 (62%) vs 291 (71%; p = 0.0067), with AML transformations in 83 (18%) vs 84 (21%; p = 0.2055) patients, respectively. Conclusions Truncating ASXL1 mutations, along with ASXL1-mutation weighted CMML prognostic models failed to effectively risk stratify pCMML patients. In pCMML, male gender and AMC ≥ 10 x 10 9/L, effectively risk stratified for OS, while these factors along with PB blasts ≥ 5% effectively risk stratified for LFS.

Five-Year Real World Data on AML Retrospective Study from Public Health Center in Brazil

Introduction: Acute myeloid leukemia (AML) is a heterogeneous aggressive leukemia with poor prognosis. The standard remission induction regimen for medically eligible patients with AML consists of a backbone of cytarabine & an anthracycline (“7+3” therapy). Brazil is a large country with striking differences in in climate, ethnic heterogeneity and socioeconomic factors. Objectives: To assess effectiveness (Eligible to the Next Treatment-ENT CR and suficient performance status to the next treatment, PFS and OS) and security (Early Mortality) of cytarabine and anthracycline in a public health center in Salvador, Brazil. Subgroups analyzed were about FTL3 and NPM1 mutations, leukocytes (>10,000 or <10,000), platelets (>20,000 or <20,000), and transplanted or non-transplanted. Methods: It was a retrospective analysis of all the cases of non-promyelocytic AML diagnosed between 2018 and 2022 in the University Hospital Professor Edgar Santos of Federal University of Bahia, after being approved by the Ethics Committee of the hospital. Kaplan-Meier methods were used to determine the median of the time to OS and PFS. Results: A total of 45 patients were analyzed. Median age at diagnosis was 43 years (range, 16-69 years) with 62% females. There were 21% and 11% patients with FLT3-ITD and NPM1 mutations, respectively. Only 14 patients had karyotype test (85% with normal karyotype). Regarding effectiveness, 52% of patients were eligible for the next therapy after complete remission (consolidation or stem cell transplantation). Refractory patients were 20%. Early (4 weeks) mortality was 28% (19% in <40a, 27% in 40-55a and 40% in >55a). The median of PFS and OS were 3.6 and 8.2 months respectively. Patients with FLT3 mutation, platelet < 20.000 and leukocytes < 10.000 had poor outcomes. In AML patients after stem cell transplantation, PFS and OS were, respectively 20 and 43 months. Average waiting time for start induction therapy was 12 days. Conclusion: The outcomes were consistent with the literature, adjusted for the population in question. Even with access to diagnostic tests (not common in Brazilian's health public centers), patients did not have access to targeted therapies, with the 7+3 regimen being the only treatment for fit patients. To achieve complete remission followed consolidation/transplantation is still the BEST scenarios in AML eligible patients, but the improvement of access to diagnostic and treatment is still an unmet need.

Azacitidine Plus Venetoclax Maintenance in Acute Myeloid Leukemia Produces Sustained Remissions with Low Toxicity: Results of a Phase 2 Study

Background: Most patients diagnosed with acute myeloid leukemia (AML) achieve remission following induction therapy. However, disease relapse remains a major clinical concern. The QUAZAR AML-001 study demonstrated improved overall and relapse-free survival with oral azacitidine (CC-486) maintenance. We designed a phase 2 study (NCT04062266) to evaluate low doses of azacitidine plus venetoclax as maintenance therapy following both intensive and low-intensity induction in AML. We present updated results of this study with close to 2 years of median follow-up. Methods: Patients ≥ 18 years of age with non-APL AML in first complete remission (CR) or CR with incomplete count recovery (CRi) not immediately eligible for allogeneic stem cell transplantation (SCT) were eligible. Cohort 1 enrolled patients who had received at least 2 cycles of intensive therapy (defined as containing intermediate to high-dose cytarabine). Cohort 2 enrolled patients who had received at least 3 cycles of low-intensity therapy (defined as hypomethylating agent or low-dose cytarabine-based). Patients with positive measurable residual disease (MRD) by flow cytometry were eligible in CR1 or beyond. Other inclusion requirements were ECOG ≤ 3, adequate hepatic/renal function, and adequate bone marrow reserve (defined as absolute neutrophil count > 0.5 x 10 9/L and platelets > 30 x 10 9/L). Treatment consisted of azacitidine 50 mg/m 2 SQ/IV on D1-5 and venetoclax 400 mg PO (adjusted for concomitant CYP3A inhibitors) on D1-14 in 28-day cycles, up to 24 cycles. Venetoclax duration could be reduced to D1-7 per physician discretion to mitigate myelosuppression and dose modifications during subsequent cycles were allowed. Antibacterial, antifungal, and antiviral prophylaxis were recommended in all patients. The primary objective was relapse-free survival (RFS, defined as time from enrollment to progression or death). Secondary objectives included overall survival (OS), safety/toxicity, and MRD clearance. Patients subsequently becoming eligible for SCT were taken off study and censored for analyses at the time of SCT. Results: 35 patients have been enrolled (25 in cohort 1 and 10 in cohort 2). The median age was 47 years in cohort 1 and 71 years in cohort 2. The baseline characteristics are shown in table 1. The median number of cycles given was 9 (range 1-24). 22/35 (63%) patients had 7 days of venetoclax during cycle 1. Median follow-up is currently 20.9 months (m). For the full cohort, the median RFS was not reached (NR; 2-year 60%) and the median OS was NR (2-year 75%). In cohort 1, the median RFS and OS were NR (2-year 71%) and NR (2-year 77%), respectively. In cohort 2, the median RFS and OS were 23.5 m (2-year 34%) and NR (2-year 67%), respectively. When stratified by ELN 2017, patients who were favorable (n=16), intermediate (n=11), and adverse (n=8) had a median RFS of NR (2-year 85%), NR (2-year 64%), and 4.0 m (2-year 0%), respectively. Corresponding median OS were NR (2-year 91%), NR (2-year 76%), and 14.9 m (2-year 27%), respectively. 4/8 (50%) of the ELN adverse patients had complex cytogenetics and/or TP53 mutations. Patients who were MRD-positive at enrollment (n=7, 3 with prior venetoclax exposure) had significantly shorter RFS compared to those who were MRD-negative (median 4.0 m versus NR, p<0.01). Only 2/7 (29%) MRD-positive patients converted to MRD-negative while on maintenance. As mutations in NPM1, IDH1, or IDH2 may sensitize AML to venetoclax, we analyzed RFS based on the presence or absence of these mutations (figure 1). Patients with sensitizing mutations had a trend towards longer RFS (median RFS NR versus 23.5 m, 2-year RFS 79% vs 36%, p=0.08). The most common grade 3/4 adverse events were infections (26%), neutropenia (17%), thrombocytopenia (17%), hypotension (6%), and neutropenic fever (6%). 4 (11%) patients required cycle 2 venetoclax dose reductions. 8 (23%) of patients went off protocol to receive SCT. All observed deaths (n=8) occurred following AML relapse (n=7) or SCT-related complications (n=1). Conclusions: Lower dose azacitidine plus venetoclax is a feasible and safe maintenance strategy in AML. This approach yields encouraging results in MRD-negative patients with non-adverse ELN risk (favorable or intermediate risk). A genetic-guided strategy may help select patients who benefit the most from this combination (i.e. those with NPM1, IDH1, or IDH2 mutations).

A Clinical-Molecular Prognostic Scoring System for Myelodysplastic Syndrome in Asia - a Multicenter Study of the Asian Myeloid Working Group (AMWG)

Background: In Asia, treatment decisions in myelodysplastic neoplasms (MDS) are conventionally based on the Revised International Prognostic Scoring system (IPSS-R). A unique clinical-molecular prognostic model, the IPSS-molecular (IPSS-M), has been developed based on 2957 patients with de-novo MDS, therapy-related MDS, and MDS/myeloproliferative neoplasm (MPN) overlap syndrome. The IPSS-M comprises haematological parameters, karyotypic abnormalities and somatic mutations in a 31-gene panel. Validation of the IPSS-M in large multicentre studies in Asian has however not been performed. Hence, prognostic models taking into account the unique clinical-molecular characteristics of Asian MDS patients remain undefined. Aims: The objectives of this multicenter cohort study were: 1. to define the haematological, cytogenetic and molecular characteristics of Asian MDS patients; and 2. to develop a prognostic model based on these characteristics. Methods: Consecutive MDS patients in Hong Kong, Singapore and Taiwan diagnosed between 2004 to 2021 were studied. Data were censored on 17 September 2022. Patients with MDS/MPN overlap syndrome were excluded. A panel of 54 myeloid-related genes was sequenced in the diagnostic bone marrow by next-generation sequencing. Hematological, pathological, cytogenetic and molecular variables were evaluated for their association with overall survival (OS), leukemia-free survival (LFS), and time to progression to secondary acute myeloid leukemia (TTP-sAML). Variable selection was performed to determine the confounding variable and independent prognostic variables. The relative weights of the selected variables were estimated using a Cox multivariable model adjusted for confounders. According to the weights of each prognostic variable, individual patient-specific prognostic scores were calculated. Based on the scores of the entire cohort, six prognostic subgroups were defined. The statistical predictive power of this prognostic model was assessed by the concordance index (C-index). The prognostic model was validated with publicly available data from the IPSS-M cohort. Results: Seven hundred and seventy-eight men (63.5%) and 447 women (36.5%) at a median age of 68.4 (interquartile range, IQR: 57-77) years were studied. After a median follow-up of 2.85 (IQR: 0.97-7.06) years, there were 693 deaths (56.6%) and 284 transformations to sAML (23.2%). Three-hundred and ninety-four patients (32.2%) received hypomethylating agents (azaciticine, N=367; decitabine, N=27), and 158 patients (12.9%) underwent allogeneic haematopoietic stem cell transplantation. All patients showed at least one genetic alteration in the 54-gene panel (Figure 1A). Prognostic scoring systems (each with 6 prognostic subgroups) were established for OS (C-index: 0.72) (Figure 1A), LFS (C-index:0.71) (Figure 1B) and TTP-sAML (C-index: 0.74) (Figure 1C). Genomic factors significantly associated with inferior outcomes were monosomy 7, del(5q), and mutations in GNAS and TP53 for OS; trisomy 19, del(5q), monosomy 7, and mutations in GNAS, PTPN11 and TP53 for LFS, and i(17q), del(5q), and mutations in NPM1, NRAS, GNAS, IDH2, SF3B1, and RUNX1 for TTP-sAML. Using the IPSS-M publically available dataset as the validation cohort, our prognostic model gave C-indices of 0.70, 0.71 and 0.74 for OS, LFS and TTP-sAML respectively. Finally, this prognostic model had superior prognostic power as compared with the IPSS-R and IPSS-M for the current Asian cohort (Figure 1D). Conclusion: Combining genomic with hematological and cytogenetic parameters, the Asian clinical-molecular prognostic model improved the risk stratification of patients with MDS in Asia, potentially improving clinical decision-making.

Fatty Acid Binding Protein FABP5 Inhibition Activates Retinoic Acid Signaling and Induces Differentiation in Acute Myeloid Leukemia

Introduction. Accumulation of undifferentiated immature blood cells is a hallmark of myeloid neoplasia (MN), and indicates decoupling of terminal-differentiation from proliferation. Therefore, reinstating to a programmed state of differentiation (lineage-maturation) is a treatment approach, exemplified by all-trans retinoic acid (ATRA) treatment of acute promyelocytic leukemias that contain t(15;17) (q24;q21). This translocation inactivates retinoic acid receptor alpha (RARA) due to its fusion with promyelocytic leukemia PML resulting in PML-RARA. This fusion gene responsible for cellular transformation confers sensitivity to treatment with ATRA. However, ATRA's therapeutic-success in APLs has not extended to other AML phenotypes. We and others have shown that RA is a ligand, not just for RARs, but also pro-survival peroxisome proliferator-activated receptor β/δ (PPARβ/δ), a member of the nuclear hormone receptor superfamily transcription factors that regulate several metabolic pathways. RA is transported by two carrier proteins, cellular retinoid-binding protein CRABP2, or fatty-acid-binding protein 5 (FABP5), depending on cellular context. These carriers preferentially deliver RA to RARs versus PPARβ/δ respectively. Somatic lesions such as loss of function TET2 or Tp53 mutation, NPM1 and ASXL1 mutations, are associated with upregulated FABP5 and downregulated CRABP2. In AML cells with these mutations, therefore, the high FABP5 can be expected to deliver RA to PPARβ/δ instead of RAR, to potentially contribute to decoupling of differentiation from proliferation. We therefore investigated this possibility and its treatment implications. Methods. Overexpression, gene knockdown/knockout (ko/kd) of key regulators of RAR and PPARβ/δ were generated in specific genotypes of AMLs. Small molecule inhibitors of FABP5 were used to further interrogate the pathways in a translationally relevant manner, both in vitro, in both malignant and normal cells, and in pre-clinical in vivo models. Results. Gene expression analysis of AMLs in the Beat AML and TCGA databases demonstrated upregulation of pro-survival PPARβ/δ target genes including FABP5 and downregulation of differentiation promoting RAR pathway genes including CRABP2in AMLs 1. In particular, FABP5 was highly expressed in AMLs containing TET2 NPM1, TP53 and ASXL1 mutations. AML cell lines OCIAML3 (NPM1 ins/DNMT3a R882c), OCIAML5 (ASXL1 Y591*/TET2 S825*), THP1 (TP53 del) and isogenic CRISPR engineered K562 (TET2 +/+/TET2 -/-) demonstrated 4-100 fold higher FABP5 expression compared to normal myeloid precursors. These models did not differentiate in response to pharmacologic concentrations of ATRA. To test if FABP5 is necessary and sufficient to maintain the undifferentiated state of these cells in the presence of ATRA, we genetically inactivated FABP5 either using RNAi (kd) or CRISPR (ko). FABP5 knockdown or knockout cells created sensitivity to ATRA, resulting in terminal-differentiation as observed by flow cytometry for terminal-differentiation markers ( See Figure). FABP5 was then inhibited using highly specific small molecule FABP5 inhibitors with a 56-fold higher affinity compared to ATRA 1: combination of iFABP5 and ATRA induced terminal-differentiation with significant upregulation of the monocyte lineage markers CD14 and granulocyte lineage marker CD11b measured by flow cytometry at 72h. Toinvestigatewhether knocking down FABP5 and ATRA treatment affects gene expression changes we performed transcriptomic profiling of OCIMAL3 Scr, shFABP5 cells treated with or without ATRA using whole exome RNA sequencing. Treatment of FABP5 kd OCIMAL3 cells with ATRA resulted in the upregulation of RAR pathways genes and downregulation of proliferation promoting genes. Conclusion. In sum, recoupling of lineage-maturation to proliferation can be achieved in non-APL AMLs that highly express the RA carrier FABP5, by inhibiting FABP5 using small molecules. This candidate treatment modality is distinct from cytotoxic chemotherapy, and importantly, can spare normal hematopoiesis.

Measurable Residual IDH2 before Allogeneic Transplant for Acute Myeloid Leukemia

Introduction: Detection of measurable residual disease (MRD) in AML patients during remission is associated with adverse clinical outcomes after allogeneic hematopoietic cell transplant (alloHCT), including recent strong evidence from our group regarding residual NPM1 and/or FLT3-ITD variants (PMID: 36881031). Large studies examining the significance of other potential MRD targets, including isocitrate dehydrogenase isoform 2 ( IDH2), are needed. IDH2, which is mutated in approximately 12% of AML patients, is of particular interest due to the possibility of targeted clinical intervention. Methods: Patients over the age of 18 in first complete remission (CR1) with IDH2 mutated AML undergoing alloHCT transplant at a CIBMTR site between 2013-2019 with remission blood samples within 100 days of alloHCT and available clinical data were included in this study. Residual variants in IDH2, NPM1, and FLT3genes were detected by ultrasensitive error-corrected next-generation sequencing (NGS-MRD) on genomic DNA from pre-conditioning blood and validated by digital droplet PCR (ddPCR). The primary outcomes were overall survival (OS) and cumulative incidence of relapse with non-relapse mortality as a competing risk. The day of transplant was considered as time 0, and the median follow-up time was calculated for censored patients. Kaplan-Meier estimation (log-rank tests), Cox proportional hazards models, and Fine-Gray regression models were fitted to examine the clinical outcomes. Results: Among the 257 IDH2-mutated AML patients in this study, 57 (23%) experienced relapse, with 41 events occurring within 12 months after alloHCT. The median follow-up time was 25 months. Among 247 patients with pre-transplant remission flow cytometry results reported, 25% were positive, but no difference in rates of relapse or OS was observed between positive and negative patients. NGS MRD analysis detected residual IDH2 variants in remission samples of 130 patients (51%) with variant allele fraction (VAF) ranging from 0.05-56% (median: 3%), and 100% validation by ddPCR. NGS MRD IDH2 positive patients had increased rates of relapse (p = 0.03; 3yr: +11%, 95% CI: 0.2% to 22%) and decreased OS (p < 0.001; 3yr: -25%, 95% CI: -13% to -38%) compared to those who tested negative (Figure 1A). These OS differences were still observed between NGS MRD IDH2 positive and negative patients, regardless of age stratification (under 60yr p < 0.001, 3yr: -28%, 95% CI: -12% to -43%; over 60yr p = 0.04, 3yr: -19%, 95% CI: -1% to -40%); however, only patients over 60yrs had increased rates of relapse (p = 0.007; 3yr: +21, 95% CI: 6% to 36%). The impact of co-mutation status was examined by subdividing the cohort based on the presence of NPM1 or FLT3-ITD mutations at baseline. Residual IDH2 variants in patients without baseline NPM1 or FLT3-ITD co-mutations (n = 151) were associated with decreased OS and increased relapse ( IDH2+ vs IDH2-; OS p = 0.005, 3yr: -23%, 95% CI: -6% to -40%; Relapse p = 0.01, 3yr: +19, 95% CI: 6% to 32%; Figure 1B). In contrast, residual IDH2 variants were not predictive of relapse in co-mutated patients (n=106, p = 0.5). In these patients, the only prognostic marker for relapse was NPM1 and/or FLT3-ITD MRD, with similar relapse risk between IDH2 only positive and MRD negative groups (p < 0.001; 3yr: 83% vs. 11% vs. 15%; Figure 1B). In multivariable analysis, IDH2 MRD positivity was associated with decreased OS (HR 2.5, 95% CI: 1.5-4.2, p < 0.001) and increased rates of relapse (HR 2.1, 95% CI: 1.4-5.7, p = 0.003) after adjusting for other characteristics. Conclusion: In this study, we demonstrate that adult patients with IDH2-mutated AML who have IDH2 variants detected in CR1 prior to first alloHCT experienced increased relapse rates and decreased overall survival. However, this association is significant only in patients without baseline NPM1 and FLT3-ITD mutations. In co-mutated patients, NPM1 and/or FLT3-ITD MRD serve as superior prognostic markers. Subgroup analyses show the potential influence of factors such as conditioning intensity and age on clinical outcomes. Nevertheless, larger studies with balanced subgroups and more comprehensive baseline mutational information, including IDH2 mutation subtype, are needed to better define the clinical relevance of IDH2 MRD in this context.

Updated Results from a Phase I Dose Escalation Study of the Rapidly-Switchable Universal CAR-T Therapy UniCAR-T-CD123 in Relapsed/Refractory AML

Introduction CAR-T efficacy in AML has been limited by a lack of ideal target antigens as well as the aggressive and resistant nature of disease. CD123 expression on leukemic blasts has been observed in about 80% of AML patients. However, the antigen is also expressed on non-malignant hematopoietic cells including neutrophils and endothelial cells. As consequence, targeting of CD123 with conventional CAR-T has been shown to result in persistent cytopenias, capillary leakage syndrome, CRS, and ICANS. AVC-101 (UniCAR02-T-CD123) is an adapter CAR-T, consisting of a universal CAR-T cell (UniCAR-T) and a CD123 targeting module (TM). The TM binds to the CAR-T cell via a peptide tag and induces T cell activity against CD123 bearing cells. Due to the short half-life of the TM, interruption of its continuous IV administration will rapidly switch T cell activity off, and mitigate toxicities as shown previously. Since the presentation of initial results, the study was amended to include a stricter and prolonged schedule of TM consolidation cycles. Given the excellent safety enabled by the switchable mechanism, additional dose levels (DL) of TM were added. Methods Key inclusion criteria included rrAML patients with ECOG ≤ 1, CD123 expression in ≥ 20% of blasts, and no suitable approved treatment option available or MRD positive AML patients. Initial dosing was applied using an adaptive Bayesian optimal interval design for drug-combination trials, escalating either cell dose or TM concentration based on posterior probability calculation of AEs. Dose escalation for the two additional dose levels was guided by a classical 3+3 design. Patients received standard Flu/Cy lymphodepletion (LD) for 3 days and TM was administered as continuous infusion, whereas UniCAR-T cells were given as one-time infusion on cycle 1 day 1. After completion of the initial 20-day induction cycle, responding patients were allowed to receive up to 3 consolidation cycles of 12 days continuous TM infusion with 7-14 days treatment free intervals in between cycles. Results As of this time 19 patients have received the induction cycle, 8 patients have also received at least one consolidation cycle (1-3, total 13 cycles). Patients were heavily pretreated with a median of 4 (2-7) prior treatment lines. Twelve of these patients had received a prior allogeneic (Allo) stem cell transplantation (SCT). Most of the patients (60%) were in adverse risk category according to ELN classification. In the initial escalation phase, 19 patients were treated in 16 dose cohorts. We observed a single DLT at DL 8 (reversible drop in fibrinogen levels, reported earlier) but no DLTs occurred in 11 patients treated in the yet highest dose cohort of 4 mg/day TM and 500 million UniCAR-T. Treatment was generally well tolerated irrespective of age and prior treatments. CRS was observed in 12 patients, mostly grade 1 or 2. Three grade 3 CRS and 1 grade 2 ICANS were reported, all resolving within 24 hrs from interruption of TM administration. Treatment-related AEs were most frequent in cycle 1, none were reported in cycles 3 and 4. No treatment-induced lasting myelosuppression was observed, and no patient required SCT support for WBC reconstitution. ORR in the first 19 patients was 53% (8/15) for the rrAML population and 75% (3/4) for the MRD population. Since the last presentation, extended administration of TM (20-day induction followed by three 12-day consolidation cycles 1-2 weeks apart) initiated by protocol amendment resulted in durable, ongoing responses approaching 5 months, longer than any prior. A 64-year-old MRD+ patient (#19) with 4 prior lines of therapy experienced a 3-log reduction in MRD levels (NPM1 mutation by qPCR) in both bone marrow and blood (Figure 1) and became transplant eligible. Re-administration of TM resulted in a significant re-expansion of UniCAR-T (Figure 2) without further cell dosing or LD. Another patient (#20) with overt hematological relapse after AlloSCT achieved a CR, ongoing at 5 months. Full safety and efficacy results of the additional 2 dose cohorts with prolonged TM consolidation will be presented at the meeting. Conclusions AVC-101 continues to be safe and tolerable. Repeated dosing of TM in consolidation cycles resulted in robust re-expansion of UniCAR-T with deeper remissions of extended durability. The switchable mechanism provides proven rapid reversal of safety events, enabling higher dose levels.

How Much Have Post-Transplant Outcomes Improved Since 2000 for Older Patients with Acute Myeloid Leukemia? a Study from the EBMT Acute Leukemia Working Party

Acute myeloid leukemia (AML) is a disease of older patients with a median age at diagnosis of 68 years. Patients older than 65 years historically have performed poorly as not being treated with intensive chemotherapy. Biological age is not the only culprit as other factors also play a role in reducing survival, such as performance status, the presence of comorbid conditions, and most importantly the biology of AML that is usually associated with high risk cytogenetic or molecular aberrations. Despite the increasing use of targeted therapies, allogeneic hematopoietic cell transplantation (allo-HCT) remains an important and potentially curative treatment modality for patients with AML. Over time, significant progress in allo-HCT has resulted in decreased non-relapse mortality (NRM) and allowed the delivery of allo-HCT to older patients. However, little information is available about the global impact of the current standard of care for older AML patients after allo-HCT and about the predictive factors for post-transplant outcomes. To address these challenges, we assessed real-world changes over time in transplant characteristics and post-transplant outcomes in older patients with AML (> 65 years), using a large dataset from the European Society for Blood and Marrow Transplantation registry. We identified 7215 adult patients (40% female; median age 68 years, range 65-80) with AML (secondary AML in 25%) allografted between 2000 and 2021 from a matched sibling donor (MSD; 23%), unrelated donor (UD; 63%) or haploidentical donor (Haplo; 14%). Karyotype risk was intermediate in 71% and adverse in 25%. NPM1 mutation and FLT3 ITD were present in 37% and 30% of patients, respectively.At transplant, 64% of patients were in first complete remission (CR1), 14% in CR2+ and 22% had active disease. Comorbidity index was zero in 43% of patients, 1-2 in 26% and >3 in 31%, while the Karnofsky score was <90 in 35%. Conditioning was reduced intensity (RIC) in 82% of patients. In vivo T cell depletion (TCD) and peripheral blood stem cells (PBSCs) were given to 62% and 93% of patients, respectively. Median follow up calculated by reverse Kaplan Meier was 40 months (interquartile range [IQR] 38-42). We compared changes in patient and transplant characteristics over time in 728 (10%) patients transplanted in 2000-2009, 1775 (25%) patients transplanted in 2010-2014, and 4712 (65%) patients transplanted in 2015-2021. Patients transplanted in recent years (65%) were older, were more likely to be in CR1 and to have an adverse-risk karyotype. Patients transplanted more recently were less likely to have had an MSD but more likely to have had a Haplo, PBSCs and TCD. The 3-year cumulative incidence of relapse (CIR) gradually and significantly decreased from 37% to 31%, then to 30% over the 3 time periods (p=0.001) whereas NRM significantly decreased from 31% and 31% to 27% (p=0.003). The 2-year leukemia free survival (LFS) and overall survival (OS) gradually and significantly improved over time from 32% to 38%, and then to 44% (p=0.001) and from 37% to 42%, and then to 49% (p=0.001), respectively, whereas graft-versus-host disease (GVHD)-free, relapse-free survival (GRFS) improved from 22% to 29%, and then to 34% (p=0.001). In a Cox regression multivariate analysis (MVA), a significant reduction in CIR and increased LFS, OS and GRFS were noted after 2015. LFS, OS and GRFS were also negatively affected by secondary AML, low Karnofsky score, active disease at transplant, and adverse-risk cytogenetics. GRFS was also negatively affected by older age, and positively affected by TCD. RIC positively affected OS whereas PBSCs positively affected LFS and OS. This improvement of post-transplant outcomes over time was observed for patients in CR1 with LFS and OS increasing from 39% to 44% and then to 47% (p=0.005) and from 43% to 48%, and then to 52% (p=0.006) as well as for patients in CR2 with LFS and OS increasing from 29% to 40% and then to 47% (p=0.001) and from 37% to 44% and then to 52% (p= 0.001), respectively. In conclusion, in older patients with AML, we observed an impressive improvement over time in post-transplant outcomes with decreased CIR and improved LFS, OS and GRFS. These large-scale, real-world data can serve as a benchmark for future studies in this setting and indicate that the opportunity for transplant for the elderly should be mandatory and no longer an option.

Genomic Characterization of Newly Diagnosed Acute Myeloid Leukemia in Patients Age 60 Years and Older; A Report from the Beat AML Master Trial

Background. Acute myeloid leukemia (AML) is a heterogenous disease that occurs primarily in older adults with a median age of 69 years at diagnosis. Most studies that characterize the genomic landscape of AML include younger adults with the majority of patients being younger than 60 years of age. We aimed to study the mutational landscape of newly diagnosed AML patients aged 60 years or older. Methods.Samples were collected from the multi-center Beat AML Master Trial (Burd, Nat Med 2020). Patients were enrolled between 2016 and 2023. Informed consent was obtained according to the Declaration of Helsinki. Patients diagnosed with non-APL AML and who were aged ≥ 60 years old were included. Patients underwent bone marrow aspiration and biopsy, cytogenetic analysis, FLT3-ITD ratio assessment (LeukoStrat CDx FLT3 Mutation Assay, Invivoscribe) and next generation sequencing (NGS) using FoundationOne®Heme(Foundation Medicine) to interrogate the entire coding region of 406 genes and select introns of 31 genes involved in rearrangements, as well as RNA sequencing to interrogate 265 genes known to be somatically altered in human hematological malignancies. The presence of multiple hotspot mutations within one gene in an individual patient was counted as one gene mutation. Patterns of co-occurrence between gene mutations were calculated using the Phi correlation coefficient. Statistical analyses were performed using R statistical software. P<0.01 was considered statistically significant. Results. A total of 1088 newly diagnosed AML patients were identified, of whom 1032 were 60 years or older. Patients had a median age of 72 years at time of diagnosis (range 60-92 years). Forty-two percent of the patients were female, most patients were non-Hispanic and 79% were Caucasian. NGS data were available for 1024 patients and mutations were identified in 461 different genes. Patients had a median number of 11.0 mutations (range 3.0 to 28.0). The most frequently mutated genes were DNMT3A (n=257; 25.1%), TP53(n=255; 24.9%), TET2 (n=243; 23.7%), RUNX1 (n=225; 22.0%), SRSF2 (n=221; 21.6%), ASXL1 (n=216; 21.1%), NPM1 (n=206; 20.1%), FLT3 (n=205; 20.0%), IDH2 (n=190; 18.6%), NRAS (n=208; 16.9%), PTPN11(n=104; 10.2%), STAG2 (n=101, 9.9% and IDH1 (n=99; 9.7%) (Figure 1A). FLT3-ITD was present in 98 (12.1%) of the 810 patients who were tested. Strongest significant co-occurrence was found between FLT3and NPM1 (n=103), STAG2 and ASXL1 (n=63), ASXL1 and RUNX1 (n=92), ASXL1 and SRSF2 (n=88), RUNX1 and SRSF2 (n=94), STAG2 and SRSF2 (n=51) and DNMT3A and NPM1 (n=92) (p<0.01). Pairwise negative correlations were observed between TP53 and NPM1 (n=6), TP53 and SRSF2 (n=17), and between NPM1 and RUNX1 (n=6) (p<0.01) (Figure 1B). The cytogenetic profiles and the full genomic landscape will be presented during the ASH meeting. Discussion. This is the largest study investigating the mutational profile in older adults with AML aged 60 years and older. Due to the 7-day screening period of the Beat AML study, patient selection was biased towards less-rapidly proliferative AML. We showed that the pattern of gene mutations was markedly different from other studies in AML that mainly included younger AML patients (Papaemmanuil, NEJM 2016; TCGA, NEJM 2013 ). The frequency of mutated TP53 was substantially higher in older AML patients, as was the occurrence of myelodysplasia-related gene mutations (e.g., SRSF2, RUNX1, ASXL1, STAG2, U2AF1, and SF3B1) and gene mutations in IDH1, IDH2 and TET2. In contrast, NPM1 mutation was less frequently mutated in older AML patients. Furthermore, we demonstrated that a large proportion (46%) of the AML patients older than 60 years of age do have a mutation that is potentially targetable for treatment ( FLT3 IDH1 IDH2 and NPM1). The high incidence of mutated TP53 and MDS-related gene mutations underscores the increased incidence of high-risk AML and the need for innovative personalized therapies in this older age group. Figure 1. A. Co-occurrence of the 15 most frequently mutated genes, and B. co-correlation between the 15 most frequently mutated genes. Blue indicates positive correlation, red indicated negative correlation. Only significant correlations (p<0.01) are shown.

Phase 1/2 First-in-Human Study of the Menin-MLL Inhibitor DSP-5336 in Patients with Relapsed or Refractory Acute Leukemia

Introduction: Menin inhibitors are an exciting new class of agents in development for patients (pts) with acute leukemia who overexpress HOXA9 and MEIS1 genes. Although there is a growing list of fusion genes in acute leukemia that appear to be dependent on homeobox-related gene activity, menin inhibition has been shown to be particularly active in pts with NPM1 mutations (NPM1c) and KMT2A rearrangements (MLLr). DSP-5336 is an investigational, oral small molecule designed to inhibit the menin and MLL protein interaction. Methods: A Phase 1/2 study of DSP-5336 is being conducted in pts with relapsed or refractory (R/R) acute leukemia. The dose escalation portion of the study consists of two parallel arms (Arm A: without concomitant anti-fungal azole therapy; Arm B: with concomitant azole therapy). Pts were eligible with R/R AML, ALL or acute leukemia of ambiguous lineage without a limit on number of prior therapies, with a focus on those with MLLr and NPM1c. Results: Accrual is ongoing with 24 pts enrolled as of April 2023; 14 pts in Arm A at doses of 40 mg BID (n=2), 80 mg BID (n=4), 100 mg BID (n=2) and 120 mg BID (n=6), and 10 pts in Arm B at doses of 40 mg BID (n=4) and 60 mg BID (n=6). The median age was 67.0 years (range 20 - 85) and 62.5% were male. All pts had AML except for 1 pt with acute leukemia of ambiguous lineage. Pts had received a median of 3 (range 1 to 9) prior treatments, and 6 (25%) had received prior alloSCT. MLLr or NPM1c were present in 6 (25%) and 4 (17%) patients, respectively. No dose-limiting toxicities have been observed thus far. Most treatment-emergent adverse events (TEAEs) were grade 1/2. TEAEs assessed as related to DSP-5336 in ≥ 3 pts were nausea (Grade 1 or 2, n = 3 pts) and vomiting (Grade 1 or 2, n = 4 pts). Grade >3 TEAEs occurring in ≥ 3 pts included anemia (6 pts), pneumonia (5 pts), sepsis (3 pts) and hypokalemia (3 pts), all unrelated to study drug. A possible Grade 4 differentiation syndrome (DS) was observed in 1 pt with a TP53 mutation in Arm A receiving 80 mg BID although concurrent pneumonia and sepsis made a clear attribution difficult. No other DS events of any grade have been reported to date. No cardiac toxicities, and no QTc prolongation of any grade related to DSP-5336 have been observed. Out of the 6 pts enrolled with MLLr, 4 were treated at dose levels projected to be active based on preclinical modeling (≥ 60 mg BID with azoles, or ≥ 120 mg BID without azoles). Of these 4 pts, all had received prior intensive induction chemotherapy as well as a venetoclax-based regimen, and 3 had received prior allo-transplant. Of these 4 pts, 1 achieved CRi with a duration of therapy of 5.1 months, 1 achieved MLFS with a duration of therapy of 6.2 months (ongoing), and 1 achieved SD with clearance of peripheral blasts, recovery of peripheral counts, resolution of leukemic gingival infiltration, and reduction in bone marrow (BM) blasts from 85% to 31%. Of the 4 enrolled pts with NPM1c, 2 were treated at doses projected to be effective. Stable disease with complete clearance of peripheral blasts were observed in both pts, and BM blasts were reduced by 66% and 83% respectively. Preliminary pharmacokinetic (PK) analysis has demonstrated generally higher mean exposures at higher dose levels. Across all the dose levels, plasma t 1/2 ranged from 2-6 hours in Arm A and from 2-10 hours in Arm B, with T max reached within 2 hours. Little to no drug accumulation was observed with repeat dosing in both arms. Data to date suggests that azoles may not have a significant effect on DSP-5336 exposure, but enrollment and comparative analyses are ongoing. Target pharmacodynamic changes have been achieved in pts who have MLLr or NPM1m, including rapid decreases in HOXA9, MEIS1, and PBX3 with treatment and, conversely, increases in CD11b compared to pretreatment. Conclusion: DSP-5336 has been well tolerated with no DLTs to date in heavily pretreated R/R AML patients with NPM1c and MLLr AML. Importantly, no cardiac signals (including no QTcF prolongation) have been observed. PK studies have not identified a significant drug-drug interaction with azoles. Although there are early signs of clinical activity and pharmacodynamic changes during administration of DSP-5336, the study is ongoing to determine a recommended Phase 2 dose for single-agent expansion and potential combination regimens. Updated safety and efficacy data will be presented at the meeting.

Decoding the Epigenetic Drivers of Menin-MLL Inhibitor Resistance in KMT2A-Rearranged Acute Myeloid Leukemia

Menin inhibitors that disrupt Menin-MLL interaction are candidate therapeutic agents for several acute myeloid leukemia (AML) subtypes such as KMT2A rearrangement and NPM1 mutation. Several Menin inhibitors are currently being assessed in clinical trials and have demonstrated promising initial results. However, the development of both innate and acquired resistance to Menin inhibitors could limit their therapeutic potential. While studies have found that around 40% of these resistance cases can be traced back to mutations in the Menin gene, the Menin gene remains unaltered in other majorities, suggesting the presence of other mechanisms driving resistance. Here, we developed a unique chromatin-focused CRISPR knockout library, named Advanced Catalogue of Epigenetic Regulators (ACER). The ACER library contains approximately 800 traditional and newly-predicted epigenetic regulators using epigenetic network analyses that integrated publicly available gene codependency (DepMap) and protein-protein interaction data (BioPlex) (Figure A). To understand the chromatin mechanisms responsible for Menin-MLL inhibition resistance, we employed the ACER library for CRISPR screen in the context of Menin inhibition. Notably, our screen identified non-canonical polycomb repressive complex PRC1.1 (PCGF1, BCOR, and RYBP) and PRC2.2 (EZH2, SUZ12, and EED) among the top hits that conferred resistance to MLL-Menin inhibition upon gene depletion (Figure B). Consistent with our screen results, the depletion of PRC1.1 components led to a markedly increase in IC50 values when treated with the Menin inhibitor VTP50469 in both human and murine KMT2A-rearranged cell models. While treatment with Menin inhibitors typically induces cellular differentiation in leukemia cells, depletion of PRC1.1 genes resulted in an almost complete blockage in this differentiation process. Menin inhibitors evict Menin from chromatin and repress the expression levels of Menin-MLL target genes like MEIS1, PBX3, and MEF2C. Strikingly, our ChIP-seq and RNA-seq data indicatethat the loss of PRC1.1 components doesn't impact Menin's displacement from chromatin or the repression of KMT2A target genes by the Menin inhibitor. This is consistent with recent data from phase I clinical trial of revumenib (SNDX-5613) and preclinical patient derived xenograft models, wherein the Menin inhibitor persistently inhibitedkey KMT2A targets, even in resistant samples. These findings highlightthe possibility that resistance to Menin inhibition arising from mechanisms independent of canonical KMT2A target genes. Importantly, our unbiased transcriptomic analysis revealed that MYC signatures as the most affected pathway in PRC1.1-depleted cells following Menin inhibition. Furthermore, we show that both the transcriptionally active Menin-MLL and the repressive PRC1.1 complexes coexist at the MYC promoter, and the interplay between these two complexes determines MYC transcription output in AML. Moreover, enforced expression of MYC leads to resistance against Menin inhibition, while genetic and pharmacologic targeting MYC in PRC1.1 deficient cells rescued the resistance to Menin inhibition. Finally, in our RNA-seq datasets, we observed a diminished monocyte differentiation signature after PRC1.1 depletion. Considering AML cells with monocytic signatures, typically seen in KMT2A-rearranged AML, often exhibit resistance to BCL-2 inhibitor venetoclax, we hypothesized that AML cells lacking PRC1.1 could be more susceptible to BCL2 inhibition. This was confirmed in our ACER library screen with venetoclax treatment, where the PRC1.1 components PCGF1 and BCOR stood out as top epigenetic factors rendering drug sensitivity. Our subsequent studies using murine AML models and human primary samples further validated that PRC1.1-deficient AML cells are markedly sensitive to venetoclax. Furthermore, we show that venetoclax can be employed to overcome the resistance to Menin-MLL inhibition. In summary, our study identifiesthe non-canonical PRC1.1 as a key epigenetic driver of Menin-MLL resistance through a KMT2A target gene-independent mechanism which involves aberrant activation of MYC. We have further provided evidence that AML cells with loss of PRC1.1 are hypersensitive to BCL-2 inhibitor Venetoclax, opening a new therapeutic avenue for tackling Menin-resistant AMLs driven by polycomb inactivation.

Impact of Residence in an Agricultural Zone on AML Characteristics

Background: Acute Myeloid Leukemia (AML) is associated with several well-established risk factors. These include cigarette smoking, exposure to benzene, to ionizing radiation, and previous exposure to chemotherapy. Interestingly, studies have observed higher incidence rates of AML in farming regions, suggesting that farming activities and related exposures, such as pesticides, may increase the risk. However, data regarding the potential variations in clinical, cytogenetic, and molecular profiles between individuals living in rural and urban areas are lacking. Therefore, we aim to specifically examine the characteristics and the outcomes of AML patients living in agricultural areas. Patients and methods: We performed a retrospective analysis including 315 patients newly diagnosed with AML from two French institutions, namely Amiens University Hospital and Lille University Hospital, from 2008 to 2013. We collected patient demographics, along with comprehensive data encompassing clinical and biological information, including cytogenetics and molecular data. The data was obtained from the Observatoire des Hauts-de-France, enabling a comprehensive and exhaustive review of the AML cases in this specific region. We geocoded the patients' residential addresses. This process involved linking the patient's location to the corresponding agricultural areas per municipality, by using geographical information from the French Ministry of Agriculture's statistics department (AGRESTE, https://www.agreste.agriculture.gouv.fr). This enabled us to establish a connection between the patients' locations and the specific agricultural regions they resided in. We categorized the population into terciles based on the proportion of local companies engaged in farming activities. Results: Approximately one-third (n=108) of patients were living in areas with less than 1.3% of farm companies, one-third (n=102) lived in regions with a percentage ranging from 1.3% to 6.9%, while the remaining third (n=105) lived in areas where the proportion of farm companies exceeded 6.9%. Sex ratio (p=0.7) and median age at diagnosis (60 vs 60.5 vs 58 years, p=0.5) were similar between the 3 groups. There was no difference in body mass index (BMI), smoking activity, alcohol consumption and performance status score between the three groups. At baseline, there was no difference in blood counts (hemoglobin, platelets, leukocytes, and circulating blasts levels), bone marrow blasts percentage, AML type (de novo vs. post MDS or MPN), or WHO classification between the 3 groups. Regarding cytogenetic abnormalities, people living on agricultural lands had fewer normal karyotypes (27% vs 33% and 43%, p=0.045) and more MLL rearrangement (20% vs 8% and 5%, p=0.013). However, there was no difference in the SWOG and ELN 2017 classifications. Regarding molecular characteristics, NPM1 mutations were found less frequently in people living in agricultural regions (13% vs 30% and 29%, p=0.021). There was no difference for FLT3-ITD positivity and CEBPA mutation. There was no difference in complete remission rate and hematopoietic stem cell transplantation treatment though we could underline an especially low rate of HSCT in the < 1.3% group. OS was also similar between the three groups (Table 1.). Conclusions: Our study showed a correlation between living in agricultural areas and a higher prevalence of unfavorable cytogenetic and molecular features in AML patients, although clinical outcomes remain unchanged. These findings suggest that the proximity to agricultural activities could have an impact on leukemogenesis, by increasing the rate of MLL rearrangements and chromosomic abnormalities.

Comparing Cairo Bishop and Howard Criteria for Identifying Clinically Meaningful Tumor Lysis Syndrome Among Acute Myeloid Leukemia Patients Treated with Venetoclax and Hypomethylating Agents: A Single Center Experience

Introduction Venetoclax (ven) in combination with hypomethylating agents (HMA) is the standard of care for the treatment of acute myeloid leukemia (AML) in patients (pts) who are ineligible for intensive chemotherapy. The most serious complication of ven is tumor lysis syndrome (TLS). The phase 1b trial of ven and HMA in AML pts included strict prophylactic measures to prevent TLS. Reported rates of TLS in real world cohorts vary widely and prophylactic measures are not standardized. Methods Here we describe TLS prophylaxis (ppx) and incidence in a cohort of 100 consecutive pts age ≥18 years at the University of Pennsylvania who received inpatient or outpatient ven and HMA for up front treatment of AML between 1/2017-8/2022. TLS ppx was at the discretion of the treating team. TLS was retrospectively defined by Cairo Bishop and Howard Criteria (table 1). Howard TLS (H-TLS) criteria differs from Cairo Bishop TLS (CB-TLS) in that it requires two lab abnormalities be present in the same 24-hour period, uses calcium corrected for albumin and does not include ≥25% changes from baseline lab values. Results 92 pts had a white blood cell count (WBC) <25,000 cells/μL on the first day of treatment (table 1). 34 pts developed laboratory (lab) CB-TLS; 8 of these pts met criteria for clinical CB-TLS due to acute kidney injury (AKI). 13 pts developed CB-TLS prior to starting ven. While the original phase 1b trial mandated frequent lab monitoring as well as starting allopurinol ≥72 hours prior to ven and suggested a target of 2L of intravenous (IV) fluid per day starting the night prior to ven, TLS ppx was variable in our pts. 56 pts received allopurinol ≥72 hours prior to starting ven, 21 received ≥2L of IV fluid per day starting at least the day prior to ven, and 56 had TLS labs measured every 8-12 hours during the first 3 days following ven initiation. CB-TLS was associated with a higher ECOG score and baseline lactate dehydrogenase as well as FLT3 and NPM1 mutations. The CB-TLS group had a higher mean WBC count but there was no difference in the proportion of pts with WBC counts ≥25,000 cells/μL. There was a trend towards increased CB-TLS in pts with WBC counts ≥15,000 cells/μL; this reached statistical significance when comparing the percent of pts with non-spontaneous CB-TLS with WBC count ≥15,000 cells/μL to the rest of the cohort (33% vs 13%, p=0.034). To characterize TLS severity, we categorized the pts with CB-TLS into three groups: insignificant TLS, significant TLS, and those whose lab abnormalities were explained by a different pathology. We defined significant TLS as pts who required hospitalization (if started treatment outpatient) or ICU transfer, received rasburicase, developed AKI, or had treatment held due to TLS. 17 pts had insignificant TLS, 14 had significant TLS, and 3 were ultimately felt not to have TLS (table 2). We also compared pts who developed CB-TLS with those who met criteria for H-TLS. Of the 34 pts who met lab CB-TLS criteria, only 6 also met Howard criteria. All 6 of these pts had significant TLS. Of the 8 pts with significant TLS who only met Cairo Bishop criteria, 6 developed spontaneous TLS >3 days prior to starting treatment with ven that would have met H-TLS criteria at onset. Their lab abnormalities were not as severe by ≤3 days prior to ven start when the formal time frame for defining TLS begins. Discussion Other studies have shown lab TLS rates of 0-40% in AML pts receiving ven with HMA, with the majority reporting rates <10%. Our data show a higher rate of lab CB-TLS than other published cohorts. However, our data also show that most pts do not develop significant TLS despite less aggressive ppx than was used in the original ven trial and suggest that less aggressive ppx is appropriate for most pts. There was a high rate of spontaneous TLS in our cohort. Debate exists over the most appropriate WBC threshold for ven initiation; our data suggest an association of WBC count <15,000 cells/μL with lower TLS risk. Of the pts who developed significant TLS, a majority had spontaneous TLS; only 5 pts had significant TLS occur after starting ven. We also found that the Howard criteria for TLS better identify pts who will have adverse outcomes from TLS and may be a more clinically meaningful set of criteria to use in future studies.

Idarubicin Versus Daunorubicin Versus Mitoxantrone for Induction Chemotherapy in Acute Myeloid Leukemia: Patient Registration Study of Turkish Society of Hematology-Acute Myeloid Leukemia Working Group

Introduction: Standard induction chemotherapy in newly diagnosed acute myeloid leukemia (AML) includes a combination of standard-dose cytarabine and an anthracycline (idarubicin [IDA], daunorubicin [DNR], or mitoxantrone [MXR]). Anthracycline selection is still not standardized, and the impact of anthracycline selection on survival and response in patients with adverse genetic risk is of interest. The study aimed to retrospectively compare the effectiveness of induction regimens with respect to anthracycline preference. Methods: The study population was recruited from the Turkish AML Registry project database based on documentation of newly diagnosed AML and induction chemotherapy with IDA, DAU, or MXR in combination with cytarabine. Patients diagnosed before 31 August 2022 were received typical “7 + 3” induction chemotherapy containing 3 days of IDA, DAU, or MXR. Results: The study included 317 newly diagnosed AML patients (median age 51; range, 18 - 79; 55.2% male). Of the patients, IDA was administered in 266 (83.9%), DNR in 42 (13.2%), and MXR in 9 (2.8%). The three anthracycline treatment groups were similar between age, gender, type of AML by the World Health Organization (WHO), and risk classification by 2017 European LeukemiaNet (ELN) when evaluated according to the baseline disease characteristics. At the time of diagnosis, although the median white blood cell levels were 19 x 10 9/L (0.4 - 355) in the IDA, 7.8 x 10 9/L (0.7 - 357.2) in the DAU, and 5.5 x 10 9/L (0.6 - 34) in the MXR therapy group (p = 0.019); the hemoglobin and platelet levels and bone marrow blast percentage were similar between the treatment groups. FMS-like tyrosine kinase-3 internal tandem duplication ( FLT3-ITD) (p = 0.561) and Nucleophosmin 1 gene ( NPM1) (p = 0.430) mutation frequencies were also similar between treatment groups. Morphological complete remission (CR) was documented in 65% of all evaluable patients. Among the patient groups, CR was 67.7% for IDA, 50% for DNR, and 55.6% for MXR (p = 0.081). In univariate analysis (Table 1), significant predictors of overall survival (OS) were age ≥60 years (p <0.001), acute leukemias of ambiguous lineage (p <0.001), presence of disseminated intravascular coagulation (DIC) (p = 0.012), and FLT3-ITD mutation (p = 0.018); no significant interaction was noted between NPM1 mutation and OS (p = 0.059). Additionally, when idarubicin was taken as a reference, there was no meaningful relationship between anthracycline type and OS (p values for daunorubicin and mitoxantrone were 0.145 and 0.356, respectively). In multivariate analysis (Table 1), age ≥60 years (p = 0.001), acute leukemias of ambiguous lineage (p = 0.007), and the presence of DIC (p = 0.040) remained significant. After a median follow-up of 29.8 months, 32.5% relapse cases were documented and similarly distributed among IDA (33.1%), DNR (31%), and MXR (22.2%) therapy groups (p = 0.771). During the same period, allogeneic hematopoietic stem cell transplantation (AHSCT) was documented in 10.9% and 26.2% of patients treated for IDA and DNR, respectively. No patient progressed to AHSCT in the MXR arm (p <0.001). In the study, 142 (44.8%) deaths were reported, and there was a 48.5%, 26.2%, and 22.2% mortality rate between the IDA, DNR, and MXR treatment groups, respectively. There was no significant difference in OS by anthracycline type in both the study cohort and age group <60 years (Figures 1A and 1B). There was also no significant difference in OS in patients with the adverse genetic risk group and <60 years age group with adverse genetic risk, according to 2017ELN (Figures 1C and 1D). The results may have been influenced by the number of patients in the treatment groups of the retrospective study and by differences in fitness levels that needed to be measured systematically. Conclusions: We are encouraged that our findings mostly agree with previously published studies and that IDA, DNR, and MXR offer comparable survival values in the research and the younger patient groups. In the future, the ever-changing treatment environment in patient groups with targetable mutations makes choosing the optimal anthracycline for induction chemotherapy in AML much more difficult.

A First-in-Human Phase 1 Study of the Menin-KMT2A (MLL1) Inhibitor JNJ-75276617 in Adult Patients with Relapsed/Refractory Acute Leukemia Harboring KMT2A or NPM1 Alterations

Background: Relapsed/refractory (R/R)acute leukemia withalterations in KMT2A (also called MLL1; 9-15% of adult AML, 10% of ALL) or NPM1 (30% of adult AML) are often associated with poor outcomes. Pre-clinical studies demonstrated the relevance of the menin-KMT2A protein-protein interaction in sustaining leukemic cells with KMT2A and NPM1 alterations (Kuhn 2016). JNJ-75276617 is a potent and selective inhibitor of the interaction between the scaffolding protein menin and the methyltransferase KMT2A with preclinical activity in KMT2A-rearranged or NPM1-mutated leukemic cell lines and primary leukemia patient samples in vitro and in vivo (Kwon 2022). We report initial data investigating JNJ-75276617 in adult participants (pts) with R/R acute leukemia harboring KMT2A alterations (rearrangements, amplifications, or partial tandem duplications) or NPM1 mutations. Methods: 75276617ALE1001 (NCT04811560) is an ongoing Phase 1, multicenter, open-label, dose-finding study. Pts in dose escalation receive JNJ-75276617 orally on a 28-day cycle. As of 8 April 2023, multiple dose levels ≥15 mg have been explored on either a daily or twice daily (BID) dosing schedule. AEs were graded by CTCAE v5.0. Responses were investigator-assessed per ELN2017. Preliminary safety, efficacy and PD data are reported herein, with a focused review of the efficacy in higher dose levels with ≥3 pts dosed. Results: Fifty-eight pts received JNJ-75276617. The median age was 63 (range: 19-83) years; 56 pts (97%) had R/R AML and 2 (3%) had R/R ALL. The median number of prior lines of treatment was 2 (range: 1-7), including 10 (17%) pts with a prior allogeneic stem cell transplant. A KMT2A or NPM1 alteration was present in 33 (57%) and 25 (43%) pts, respectively. Thirty (52%) pts experienced ≥1 treatment-related AE (TRAE); most commonly differentiation syndrome (DS) (8 [14%]). Grade ≥3 TRAEs were observed in 17 (29%) pts; those reported in ≥2 pts were neutropenia (6 [10%]), anemia and thrombocytopenia (4 [7%] each), DS (3 [5%]), and ALT and AST increase (2 [3%] each). Dose limiting toxicities (DLTs) were observed in 5 (9%) pts, with DS (2 [3%]) as the only DLT reported in ≥2 pts. In 26 (63%) of the 41 pts with disease evaluation data, there was a reduction in bone marrow (BM) disease burden ( Figure 1). Of these, a ≥50% decrease in BM blasts was observed in 16 (39%) pts. In the highest dose level with ≥3 pts (90 mg BID; n=8), the ORR (≥PR) was 50% (n=4), with all responders ongoing ( Figure 2). These responders (2 NPM1-, 2 KMT2A-altered) achieved CR (1 pt), CRh (1 pt), and CRi (2 pts). In a review of higher dose levels with ≥3 pts (≥45 mg BID; n=20), the ORR was 40% (n=8), with 7 responders ongoing ( Figure 2). These responders (5 NPM1-, 3 KMT2A-altered) achieved CR (3 pts), CRh (1 pt), CRi (3 pts), and PR (1 pt); median (range) time to first response (≥PR) 1.81 mos (1.0-3.3; n=8); time to CR, CRh, or CRi 1.77 mos (1.0-3.3; n=7); and time to CR 2.79 mos (1.8-2.9; n=3). Across all cohorts there were 12 responders, including 1 MRD negative CR. One responder discontinued treatment for allogeneic transplant; however, 8 responders continue on treatment, including 2 pts in cycle 9. Preliminary PD data from unfractionated BM and/or PBMCs in paired samples among responders (n=12) show biologic activity as indicated by reduction in expression (mean fold change from baseline calculated as [on-tx-baseline]/baseline [range]) of menin-KMT2A target genes ( MEIS1 -0.42 [-1.0-9.0]; HOXA9 -0.03 [-1.0-21.7]; FLT3 18.6 [-1.0-425]) and induction of genes associated with differentiation ( ITGAM 55.0 [-0.93-1467]; MNDA 5.9 [-1.0-83.5]). Compared to baseline, the percentage of KMT2A-altered cells or NPM1 variant allele frequency (VAF) was reduced in responders, with a decrease in KMT2A-altered cells by break-apart FISH probe from 59.2% at baseline to 8.1% post-treatment and in NPM1 VAF using a myeloid gene NGS panel from 13.1% at baseline to 2.8% post-treatment. Conclusions: Dose escalation in 75276617ALE1001 is ongoing with the RP2D(s) yet to be determined. Pts in dose expansion will receive JNJ-75276617 at the identified RP2D(s). Preliminary results of this FIH Phase 1 study demonstrate that JNJ-75276617 monotherapy has an acceptable safety profile, encouraging antileukemic activity, and emerging biologic activity consistent with the proposed mechanism of action in pts with R/R acute leukemia harboring KMT2A or NPM1 alterations.

Factors Impacting Response to Olutasidenib in Patients with mIDH1 Acute Myeloid Leukemia

Introduction. Olutasidenib is an FDA-approved, targeted therapy for IDH1-mutated (mIDH1) AML. Among 147 patients with relapsed/refractory mIDH1 AML, 51 (35%) achieved a complete response (CR) or CR with partial hematologic recovery (CRh) after treatment with olutasidenib, with a duration of response of 25.9 months. We conducted sub-analyses to better understand which factors might be predictive of response to therapy. Methods. Patients were divided into groups based on achieving an overall response (CR, CRh, CRi, PR, MLFS) or having best response of stable disease or progression. Groups were compared to determine if there were baseline patient or disease characteristics that might predict attainment of an overall response to olutasidenib. The study was not powered to statistically evaluate subpopulations. Descriptive statistics are provided, and differences of 20% or more between groups were noted. Data cutoff date is June 2021. Results. As of data cutoff, 71 (48%) patients had achieved an overall response; the remaining 76 patients had stable disease (SD), clinical benefit (CB), disease progression (PD) or were not evaluated (due to short time on trial). Composite CR (CR, CRh, CRi) was achieved in 66/71 (93%) of overall responders, with PR and MLFS comprising only 3% of overall responders ( Table 1). With a median follow-up of 10.2 months (range 0.2-38.1), 20 responders were continuing treatment with ongoing responses at the time of data cutoff while 51 had discontinued; reasons for discontinuation were HSCT (n=14 all of whom had ongoing responses), disease progression (n=24), adverse event (n=8), physician decision (n=4) and protocol deviation (1). Responses to olutasidenib therapy were observed across a broad range of patient demographics and disease characteristics, most of which did not appear to impact the rate of response to olutasidenib ( Table 2). Bone marrow blast count at baseline correlated with response, with 63% of patients with baseline counts ≤40% achieving an overall response compared with 34% in those with baseline counts >40%. More patients with R132C (58%) and R132L/G/S (48%) mutations achieved an overall response compared to those with an R132H mutation (26%) However, a previous analysis showed that patients with the R132H mutation were more likely to have a FLT3 and/or NPM1 mutation, which are associated with a poor prognosis (de Botton S, et al, Blood 2021). Although fewer patients in the overall responder groups had FLT3 or NPM1 mutations compared with the non-responder group, 12/38 (32%) with NPM1 and 5/16 (31%) with FLT3 had an overall response. In addition, 2/5 (40%) patients with the TP53 co-mutation had an overall response to olutasidenib. While group outcomes for specific characteristics of disease may suggest a worse prognosis, individual patients treated with olutasidenib may achieve an overall response despite the impact of these negative factors. Two patients in this analysis had mIDH1-R132H mutations as well as FLT3 and NPM1 co-mutations and primary relapsed AML. One achieved a CRh and proceeded to transplant after a DOR of 15.5 months, and the other achieved a CRi lasting 1.3 months and then progressed. The baseline bone blast count was 53% in the first patient and 80% in the second. Two of the responders in this analysis had mIDH1-R132C and a TP53 co-mutation with refractory AML; one achieved a CR and proceeded to transplant after a DOR of 6 months, and the other had a PR and progressed after 3 months. The baseline bone blast count was 25% in the first patient and 65% in the second patient. Conclusions. This post-hoc analysis demonstrated that response to olutasidenib occurred across a wide variety of patient types and disease characteristics, including those generally associated with a poor prognosis. Baseline bone blast count was strongly predictive of response to treatment. Further evaluation of these observations is needed.

Management of Hyperleukocytosis in Pediatric Acute Myeloid Leukemia Using Immediate Chemotherapy without Leukapheresis, in Protocol NOPHO-DBH AML-2012

Introduction: Hyperleukocytosis (HL) in pediatric acute myeloid leukemia (AML) is associated with severe complications and inferior outcome. Initial management is subject to debate, and the role of invasive methods such as leukapheresis (LA) and exchange transfusion (ET) has been questioned. We report the results on HL patients included in the NOPHO-DBH AML 2012 study. Patients and methods: All patients were treated on the NOPHO-DBH AML 2012 protocol. Between January 1 st, 2013 and September 30 th, 2021, 714 patients were diagnosed, of whom 122 (17.1%) had HL, defined as white blood cell count (WBC) ≥100x10 9/L. Sixty-nine patients (9.7%) had WBC ≥200 x10 9/L. Protocol guidelines recommended immediate start of the first chemotherapy course (starting with etoposide [ETO] monotherapy for 5 days at 150 mg/m 2 once daily) followed randomized by either cytarabine/mitoxantrone (MEC) or cytarabine/daunoxome or daunorubicin (D[x]EC). The use of LA, ET, or prephase chemotherapy (PCT) was discouraged. In addition to registered data, we sent out questionnaires asking for additional detailed information on HL patients. Results: HL patients had a median WBC of 211x10 9/L (range 101-880). They did not differ from Non-HL patients in terms of age, sex or CNS involvement, but had a higher proportion of extramedullary tumors (18% vs 11%). They had a significantly lower frequency of RUNX1::RUNX1T1 fusions (1.7% vs. 15.4%), and a higher proportion of KMT2A rearrangements (except KMT2A:: MLLT3) (23.1% vs. 12.2%), and FLT3-ITD without NPM1 mutation (20.7% vs. 8.1%). Of the 122 HL patients, 111 (90.2%) were treated according to the guidelines with ETO upfront. LA was used in six patients (4.9%), in one case combined with ET. One of the LAs was performed in a patient unresponsive to ETO (increasing WBC after 2 days of ETO treatment). Eight patients received prephase chemo (various regimes) before start of the first course. In the 111 patients treated with ETO upfront, the first dose ETO was applied the same day as the AML diagnosis or the day after in 94.4%, and the drug was administered via peripheral veins in 37.8% of patients without major complications. After initiation of ETO, the remaining WBC on days 2-5 was 69.0%, 35.6%, 17.2% and 8.4% respectively, of the value before chemotherapy (Figure 1). On day 3, 80.9% had a WBC<100 x10 9/L. Compared to the non-HL group, HL patients had a trend to higher early death rate within the first 6 weeks (5/122, 4.1% vs. 13/592, 2.2%, P=0.062), and a higher rate of resistant disease defined as > 5% blasts after 2 induction courses (14/122, 11.5% vs. 23/592, 3.9%, P=0.002). Five-year event-free survival (EFS) for all HL patients was 51.9±4.8 compared to 64.7±2.1 in the non-HL group. Overall survival (OS) was 73.5±4.2 and 78.7±1.8, respectively (log rank 0.089). Three children with HL died within 2 weeks from diagnosis (2.5%). Early death within 6 weeks occurred in 3/111 (2.7%) in the ETO-upfront group and 2/11 (18.2%) in the LA/ET/PCT group, P=0.064). Of 69 patients with WBC >200 x 10 9/L, 14 patients died, but only three during the first 6 weeks. Conclusions: The NOPHO-DBH AML 2012 protocol is very effective in pediatric AML. The first chemotherapy course starts with five consecutive days of ETO monotherapy. We made use of this “ETO-window” in the management of HL patients, omitting invasive methods to reduce high WBCs. We conclude that in HL patients immediate ETO-therapy without LA/ET is feasible, easy to administer, safe and effective. Treatment results were excellent in terms of early death and OS. The significantly lower EFS for HL patients is explained mainly by a higher frequency of resistant disease, probably due to the higher proportion of high-risk AMLs (low rate of RUNX1:: RUNX1T1, high frequency of rearranged KMT2A and FLT3-ITD/NPM1wt). However, OS was not significantly lower for the HL group, which may be explained by successful salvage of patients with resistant disease or relapse using stem cell transplantation. WBC reduction during the first days of ETO therapy is comparable to reported results of LA, and outcome seems at least equivalent to studies using LA as part of their recommended therapy. Since the ETO-window allows avoiding invasive, costly and time-consuming procedures, the consortium now called NOPHO-DB-SHIP decided to stick to the same policy in the new protocol, CHIP-AML22.

Chronic Mitochondrial Dysfunction Renders Acute Myeloid Leukemia Resistant to Multipronged Inhibition of Mitochondrial Metabolism

Acute Myeloid Leukemia (AML) is a genetically diverse and aggressive myeloid neoplasm that awaits novel therapeutics to improve patient survival. Targeting AML metabolic aberrations as a genotype-agnostic strategy has shown promise, evidenced by the clinical success of venetoclax and the rapid pre-clinical development of other mitochondrial (mt) metabolism targeted therapies. However, the mechanism of how AML develops resistance to drugs targeting key mt metabolic pathways is not fully understood. We previously identified sirtuin 5 (SIRT5), a deacylase that regulates several pathways of mt metabolism in AML including oxidative phosphorylation and glutamine metabolism. In AML, it functions as a master metabolic regulator that is necessary for leukemic stem cell survival. Genetic or pharmacological inhibition of SIRT5 leads to rapid AML cell apoptosis in vitro and disease regression in multiple AML mouse models. However, a subset of AML cell lines and primary samples can survive SIRT5 inhibition, indicating an unusual intrinsic resistance to mt dysfunction. To provide a mechanistic explanation, we first compared the molecular features between SIRT5-dependent and independent cases. Among the 25 primary AML samples we tested, NPM1c mutation, but not FLT3-ITD, TP53, WT1, NRAS, or IDH1 mutations, is significantly enriched in SIRT5-independent samples. Accordingly, among the panel of 22 AML cell lines we tested, the only NPM1 mutant cell line (OCI-AML3) sits at the most SIRT5-independent extreme of the spectrum. NPM1 mutant AML cells have increased cytoplasmic mitochondrial DNA (mtDNA) and fragmented mt morphology, indicating chronic mt damage (Wu H et al. Cancer Discovery. 2021). Because SIRT5 exerts its metabolic maintenance functions within the mt, we hypothesize that AML cells that can adapt to chronic, sublethal mt dysfunction will be capable of proliferation without SIRT5. To test this, we took three orthogonal approaches: (1) introducing NPM1c mutation, (2) using low concentrations of ethidium bromide (EtBr) to selectively block mtDNA replication, or (3) genetically inducing chronic mt dysfunction by silencing the essential mt transcription factor TFAM in 3 AML cell lines that are heavily dependent on SIRT5 - OCI-AML2, CMK, and SKM-1. SIRT5-dependent cells expressing ectopic NPM1c exhibit mt dysfunction as evidenced by decreased oxygen consumption rate (OCR) measured using the Seahorse metabolic flux assay. Also, the lethal effect of SIRT5 depletion is rescued in these cells. Pharmacologically blocking NPM1c nuclear export with selinexor in the typically SIRT5-independent OCI-AML3 cells sensitizes them to the lethal effect of SIRT5 depletion. After culturing in 100 ng/mL EtBr for 3, 5, or 11 days, cells had significantly decreased mt abundance as measured by mtDNA content relative to nuclear DNA and had significantly decreased OCR and extracellular acidification rate (ECAR). SIRT5-dependent cells cultured in EtBr survived shRNA-induced SIRT5 depletion, in contrast to the rapid lethality of cells not cultured in EtBr. Additionally, cells initially cultured in EtBr and allowed to recover in the absence of EtBr assume their original phenotype of SIRT5 dependence. However, inhibiting specific subunits of the mt using potent complex I and III inhibitors (rotenone and antimycin A) shows no effect on SIRT5 dependence. Together these data suggest that mt damage, rather than loss of function in one pathway, is needed to convert SIRT5-dependent cells to SIRT5 independence. TFAM, a nuclearly coded transcription factor for mtDNA, is necessary for the replication and transcription of mt genes. We used cas13d-mediated knockdown of TFAM to deplete mtDNA and induce mt dysfunction. Experiments that investigate how this method of inducing mt dysfunction affects OCR, ECAR, and SIRT5-dependent cell line proliferation are currently underway and will be presented at the ASH conference. In summary, we present a mechanism of how NPM1 mutation-associated or experimentally induced chronic sublethal mt dysfunction renders AML resistant to therapies targeting vital mt metabolic pathways, such as SIRT5 inhibitors, demonstrating AML's metabolic flexibility. Experiments are scheduled to validate these findings in primary samples and additional metabolomic and transcriptional studies will be done to deduce the mechanism for SIRT5 dependence in AML.