Specific Acute Myeloid Leukemia Subtypes Research Articles

BRAF Mutations in Patients with Myeloid Neoplasms: A Cancer Center Multigene Next-Generation Sequencing Analysis Experience.

BRAF mutations are rare in myeloid neoplasms and are reported to be associated with poor treatment outcomes. The purpose of our study is to characterize BRAF mutations in myeloid neoplasms using a next-generation sequencing (NGS) panel based on the experiences of a single cancer center. We conducted a retrospective review of patients with myeloid neoplasms who underwent the HopeSeq studies between January 2018 and September 2023. A total of 14 patients with myeloid neoplasms carrying BRAF mutations were included in our cohort. The clinical, pathological, and molecular features of these patients were investigated. Our study indicates that BRAF mutations are rare in myeloid neoplasms, constituting only 0.53% (14/2632) of all myeloid neoplasm cases, with the most common BRAF mutation being BRAF V600E (4/14; 28.6%). Interestingly, we observed that six out of seven patients with acute myeloid leukemia (AML) exhibited AML with monocytic differentiation, and all the patients with AML exhibited an extremely poor prognosis compared to those without BRAF mutations. TET2 (5/14; 35.7%), ASXL1 (4/14; 28.6%), and JAK2 (4/14; 28.6%) were the three most frequently co-mutated genes in these patients. Moreover, we noted concurrent KMT2A gene rearrangement with BRAF mutations in three patients with AML (3/7; 42.9%). Our study suggests that although BRAF mutations are rare in myeloid neoplasms, they play a crucial role in the pathogenesis of specific AML subtypes. Furthermore, RAS pathway alterations, including BRAF mutations, are associated with KMT2A gene rearrangement in AML. However, these findings warrant further validation in larger studies.

Induction of AML cell differentiation using HOXA9/DNA binding inhibitors as a potential therapeutic option for HOXA9-dependent AML.

The mainstay of acute myeloid leukemia (AML) treatment still relies on traditional chemotherapy, with a survival rate of approximately 30% for patients under 65 years of age and as low as 5% for those beyond. This unfavorable prognosis primarily stems from frequent relapses, resistance to chemotherapy, and limited approved targeted therapies for specific AML subtypes. Around 70% of all AML cases show overexpression of the transcription factor HOXA9, which is associated with a poor prognosis, increased chemoresistance, and higher relapse rates. However, direct targeting of HOXA9 in a clinical setting has not been achieved yet. The dysregulation caused by the leukemic HOXA9 transcription factor primarily results from its binding activity to DNA, leading to differentiation blockade. Our previous investigations have identified two HOXA9/DNA binding competitors, namely DB1055 and DB818. We assessed their antileukemic effects in comparison to HOXA9 knockdown or cytarabine treatment. Using human AML cell models, DB1055 and DB818 induced in vitro cell growth reduction, death, differentiation, and common transcriptomic deregulation but did not impact human CD34+ bone marrow cells. Furthermore, DB1055 and DB818 exhibited potent antileukemic activities in a human THP-1 AML in vivo model, leading to the differentiation of monocytes into macrophages. In vitro assays also demonstrated the efficacy of DB1055 and DB818 against AML blasts from patients, with DB1055 successfully reducing leukemia burden in patient-derived xenografts in NSG immunodeficient mice. Our findings indicate that inhibiting HOXA9/DNA interaction using DNA ligands may offer a novel differentiation therapy for the future treatment of AML patients dependent on HOXA9.

Abstract 3229: IACS-16559, a CBP/P300 bromodomain inhibitor for the treatment of specific AML subsets

Abstract Block in differentiation and accumulation of undifferentiated blasts are hallmarks of Acute Myeloid Leukemia (AML) and epigenetic processes have been shown to play a critical role in hematological malignancies. Paralogs CREBBP and EP300, in association with co-factors, are hijacked during leukemogenesis by aberrant transcriptional factors, thus driving blast proliferation and maintaining the undifferentiated phenotype. Here, we introduce IACS-16559, a bromodomain inhibitor targeting CREBBP/EP300. IACS-16559 is highly selective for the bromodomains of CBP and EP300 versus BRD4, showing a dose-dependent inhibition of H3K27 acetylation residue in vivo and in vitro. In addition, IACS-16559 pharmacokinetic properties show low clearances, high oral exposures and predicted QD dosing in human. We screened AML cell lines in vitro for their response to IACS-16559 and identified specific subtypes of AML that exhibit heightened sensitivity to CBP/EP300 inhibition, specifically AML with AML1-ETO and MLL-AF9 rearrangements, and AML featuring mutations in the nucleophosmin (NPM1c) gene. IACS-16559 inhibited proliferation of AML responder cell lines in vitro, halted cell cycle progression, with an accumulation of cells in G1 and a decrease of cells in S phase. IACS-16559 did not induce apoptosis across a panel of AML cell lines, indicating that induction of apoptosis is not a major mechanism of action of CBP/EP300 inhibition. To augment the anti-tumor effects of IACS-16559, we prioritized the pharmacological combination of CBP/EP300 and MLL-Menin inhibitors. This combination synergistically impeded cell proliferation and potently triggered apoptosis in vitro and in vivo, whereas none of the individual agents alone had this effect. The changes in gene and surface marker expression produced by these treatments, as single agents and in combination with synergistic effects, were in line with an induced myeloid differentiation phenotype. Collectively, our findings provide a compelling basis for further exploring the use of a CBP/EP300 bromodomain inhibitor in conjunction with an inhibitor of the MLL-Menin interaction in a subset of AML patients with MLL rearrangements or NPM1 mutations. Citation Format: Ciro Zanca, Michael Soth, Guang Gao, Jason Gay, Ningping Feng, Christopher Bristow, Kang Le, Quanyun Xu, Phuong Nguyen, Yongying Jiang, Teresa Perry, Faika Mseeh, Paul Leonard, Jason Cross, Virginia Giuliani, Joe Marszalek, Philip Jones, Tim Heffernan. IACS-16559, a CBP/P300 bromodomain inhibitor for the treatment of specific AML subsets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3229.

Approach to Acute Myeloid Leukemia with Increased Eosinophils and Basophils

There is remarkable morphologic and genetic heterogeneity in acute myeloid leukemia (AML). In a small percentage of cases of AML, increased eosinophils and/or basophils are present in the bone marrow and sometimes in the peripheral blood. This is often a puzzling diagnostic situation but also an important finding that requires special investigation. Unique chromosomal rearrangements have been correlated with an increased number of eosinophils and basophils in AML. The identification of the underlying genetic lesion that promotes eosinophilia and basophilia can dramatically change both the prognosis and the treatment of the patient. Thus, clinicians must be vigilant in searching for the cause of eosinophilia and basophilia in patients with AML, since the different causes may lead to different treatments and survival outcomes. In this article, we examine the significance of increased eosinophils and/or basophils in the context of AML, provide guidance that simplifies the differential diagnosis, and give prognostic and therapeutic information about specific subtypes of AML associated with eosinophilia and/or basophilia. Evidence supporting personalized (molecularly targeted) therapy for these patients is also presented.

Integrated Methylation and Transcriptional Landscape and Evolution of Pediatric AML

Acute myeloid leukemia (AML) is an aggressive hematopoietic malignancy associated with poor outcomes despite intensive therapies. The genomic landscape of pediatric AML has been well characterized and has led to translational benefits, such as improved relapse predication and effective targeted therapies for a subset of individuals (e.g., FLT3-ITD AML). Despite these advances, the majority of patients continue to receive therapeutic interventions that have not changed in three decades. This underscores the need to advance our understanding of AML biology beyond genomic alterations (e.g., somatic mutations). Here, we present the largest (N = 858 patients) integrative analysis of pediatric AML paired transcriptomes and methylomes to date, performed at diagnosis for patients enrolled on Children's Oncology Group clinical trials AAML0531 (NCT00372593) or AAML1031 (NCT01371981). We also analyzed N = 400 and 253 corresponding transcriptomes and methylomes at relapse, respectively. Additionally, we performed comprehensive mutation and fusion calling from NGS data and integrated clinical covariates and outcomes. This robust dataset allowed us to uncover how pediatric AML epigenetics are perturbed from normal hematopoiesis, characterize the evolution of transcriptional and methylation changes from leukemia diagnosis to relapse, and identify more accurate relapse prediction models for pediatric AML. We initially performed unbiased hierarchical clustering on CpG sites that are differentially methylated between AML and normal bone marrow to identify distinct AML-restricted methylation (ARM) signatures. This analysis demonstrated that that these ARM clusters were enriched in specific cytomolecular subtypes of pediatric AML. Specifically, we identified methylation signatures that recapitulated their adult counterparts (e.g., IDH1/2, DNMT3A, WT1) and pediatric specific methylation signatures that clustered with RUNX1::RUNX1T1, CBFB::MYH11, KMT2A-rearrangement, NUP98::NSD1, NUP98::KDM5A, and CEBPA mutant AML. Next, we investigated the transcriptional and methylation signatures for the relapsed cases and compared them to diagnosis. We demonstrated that relapsed transcriptional and methylation signatures clustered within their cytomolecular subtype counterparts at diagnosis (i.e., AMLs at relapse more closely resemble AMLs at diagnosis with the same underlying gene fusion than other relapsed AMLs) ( Fig. 1A). However, analyzing our data in the context of these cytomolecular subtypes, we found that leukemias uniformly acquired a more stem-like state at relapse. We focused our biomarker discovery efforts on KMT2A-rearranged AMLs since (as a cohort) these patients have an intermediate risk of relapse and existing molecular biomarkers (e.g., KMT2A fusion partner and measurable residual disease detection) result in only modest improvements in relapse prediction. First, we found that CpG islands associated with CD34 and HOXA gene expression were frequently (but variably) hypermethylated, which was also associated with downregulation of CD34 and HOXA gene expression and favorable survival outcomes. Next, we performed non-negative matrix factorization on diagnosis and relapsed KMT2A-rearranged AMLs and identified a novel gene set associated with relapse and poor outcomes. These three distinct methylation-transcriptional findings (i.e., CD34, HOXA, and relapsed signatures) were subsequently transformed into a novel methylation-transcription biomarker score (MTBS) using machine learning algorithms (i.e., LASSO Cox regression). We used KMT2A-rearranged AML patients from AAML1031 as our training cohort (N = 129) to generate a MTBS that was able to distinguish KMT2A-rearranged cases with disparate outcomes. This algorithm was validated in an independent cohort from AAML0531 (N = 28) ( Fig. 1B). Additional methylation-transcriptional biomarker discovery efforts for pediatric AML are underway. Since pediatric AML is associated with a low mutational burden, understanding leukemia-specific transcriptional and epigenetic alterations is of utmost importance. Our study demonstrates the translational promise offered by deep functional genomic characterization of pediatric AML and we believe that further interrogation may provide additional novel insights into leukemia initiation, relapse prediction, and novel treatment approaches.

Iron-Related Laboratory Profile May Predict Response and MRD Negativity Rate in Acute Myeloid Leukemia Patients

Introduction: iron metabolism is deeply related to the pathogenesis and prognosis of acute myeloid leukemia (AML) and remains largely unexplored in the era of MRD-driven therapy. Several studies have assessed prognostic implications of altered iron-related laboratory values at diagnosis and there is growing interest towards the inherent pathobiology of iron in AML since new therapeutic strategies (i.e. targeting ferroptosis) are being investigated. Nonetheless, no clear association has been established between a specific risk subtype of AML within the new ELN2022 classification and a metabolic iron signature at diagnosis. Furthermore, data correlating an altered iron profile at diagnosis with post-induction response, rate of infections and need for PRBC transfusions are lacking. Methods: We performed a retrospective monocentric analysis in intensively (IC) and non-intensively (NIC) treated AML patients. Effects of altered iron parameters at diagnosis (at least one between ferritin and transferrin saturation [TSAT] according to local-lab reference range) were investigated sub-dividing patients into an altered (AC) vs a non-altered cohort (NAC). 110 AML patients diagnosed between June 2019 and April 2023 were screened for this study. 62 patients were eligible: 46 IC (AC:22 vs NAC:24) and 16 NIC (AC:12 vs NAC:4) patients were analysed. 48 patients were excluded because of previous PRBC transfusions (if > 5 PRBC), active infection at diagnosis or severe comorbidity. We compared the two cohorts in terms of ELN22 risk classification, frequency of MDS-related mutations using an NGS-based panel, TP53 mutation rate, and rate of complex karyotype (CK). Furthermore, we compared AC vs NAC (both in IC and NIC) in terms of overall survival (OS), morphologic complete response (CR) rate, rate of post-induction measurable residual disease negativity (both PCR-based and MFC based), rate of G4 infections and PRBC transfusions during induction. In addition, ferritin and TSAT cut-off values to predict CR and MRD response were calculated using ROC analysis. Selection bias was assessed considering age, risk category, PRBC count before therapy, therapy regimen, comorbidity using Charlson comorbidity index (CCI) and CRP values. Results: There was not a significant difference in terms of high risk subtype frequency between AC vs NAC group in both the IC and NIC population (IC p: 0,39/NIC p: 0,29). In both macro-groups, the AC group did not show a significantly higher frequency of CK (IC p: 0,66/NIC p: 0,84), TP53 mutations (IC p: 0,91/NIC p: 1) and MDS-related mutations (IC p: 0,76/NIC p: 0,14). OS was not significantly different in the AC group in both IC and NIC (IC p: 0,16/ NIC p: 0,62). No significant differences were found between the two cohorts in terms of post-induction CR rate (IC p: 0,6/NIC p: 0,25), MRD-negativity rate (IC p: 1/NIC p: 1) and rate of G4 infective complications (IC p: 0,5/NIC p: 0,59). In the IC patients with high risk disease, an altered iron profile at diagnosis correlated with a higher need (>10) of PRBC during induction compared to high risk patients with normal iron profile (p-value 0,014). Iron-related lab values at diagnosis were informative in predicting post-induction CR and MRD status in both the IC and NIC population. In the IC population (figure 1) a ferritin cut-off value of 190 ng/ml (AUC: 0.625) predicted CR rate while a TSAT cut-off value of 48% (AUC: 0,587) was informative about MRD negativity rate. Similarly, in the NIC population a ferritin cut-off value of 246 ng/ml (AUC: 0,772) was informative about MRD negativity rate (figure 2). Conclusions: Our retrospective study did not find an undisputable correlation between a specific ELN2022 AML risk subtype or gene-mutation using NGS and altered iron lab-values at diagnosis, whereas an association in ELN2022 high risk IC patients regarding PRBC transfused during induction in AC vs NAC was found. Although the AC did not exhibit a shorter OS compared to the NAC in both IC and NIC population, specific iron-related cut-off values were informative about disease response to both intensive and non-intensive treatments, including MRD negativity rate. Larger clinical trials to dissect iron-metabolism in AML pathogenesis, diagnosis and treatment are highly warranted. Keywords: Acute Myeloid Leukemia, iron metabolism, ferritin, transferrin saturation, ELN22, MDS-related gene mutations, NGS, induction therapy, MRD, OS, transfusion rate

Genetic Determinants of Dependency on Regulated Protein Synthesis in Leukemia Stem Cells

Acute myeloid leukemia (AML) is initiated and sustained by rare self-renewing leukemia stem cells (LSCs). LSCs do not resemble HSCs, but rather more mature hematopoietic progenitor cells that usually have no self-renewal potential. However, during leukemogenesis, LSCs become aberrantly self-renewing, but the mechanisms that allow for this are poorly understood. We recently used models of AML driven by AML-ETO9a and MLL-AF9 to show that LSCs may co-opt from HSCs a dependence on regulation of protein synthesis mediated by CD99 (ASH 2021, #2222), which we identified as a therapeutic target highly expressed in LSCs (PMID: 28123069). However, it remains unclear if LSCs from specific subtypes of AML are particularly dependent on regulated protein synthesis. In an analysis of transcriptome data from the ECOG 1900 cohort, we found that certain mutations ( KIT, PTEN, AML-ETO, IDH1/2 and FLT3-ITD) were associated with the highest levels of CD99, raising the possibility that such AML subtypes are more dependent on CD99. In the TCGA AML cohort, CD99 expression was significantly higher in AML patients with FLT3-ITD mutations but not NRAS mutations ( Fig. 1A). To test if LSCs from AMLs driven by mutations in FLT3 or NRAS differ in their dependence on regulation of protein synthesis by CD99, we crossed Cd99 -/-(CD99 KO) mice with two mouse models of AML driven by these alleles to generate Cd99 -/-; Mx1-Cre; Tet2 fl/fl; Flt3 ITD/+and Cd99 -/-; Mx1-Cre; Tet2 fl/fl; Nras LSL-G12D/+ mice (CD99 KO TET2/FLT3 and CD99 KO TET2/NRAS). TET2 was chosen as a cooperating allele given its neutral association with CD99 in the above human datasets, and it additionally co-occurs frequently with mutations in FLT3 or NRAS. To generate AML in both models, Tet2 was deleted with polyinosine-polycytidylic acid injections, followed by transplant of 4 million bone marrow (BM) cells into irradiated wild-type (WT) mice. At 16 weeks, recipients of CD99 WT TET2/FLT3 BM developed an expansion of aberrant donor-derived Mac-1 + c-kit + cells in the peripheral blood (PB), and BM aspirates revealed an accumulation of LSK CD48 +CD150 - cells that harbor LSC activity in this model ( Fig. 1B). In contrast, recipients of CD99 KO TET2/FLT3 BM exhibited significant depletion of these aberrant populations (P=0.0055 and P=0.019 respectively, n=10 for each group). Recipients of CD99 WT TET2/FLT3 BM also exhibited increased leukocytosis and thrombocytopenia, which were attenuated in recipients of CD99 KO TET2/FLT3 BM (P=0.046 and P=0.042, respectively). In contrast, there was no difference between recipients of CD99 WT TET2/NRAS and CD99 KO TET2/NRAS BM in emergence of the aberrant Mac-1 + c-kit + cells and thrombocytopenia (P=0.3237 and P=0.9752, respectively, n=10 for each group). These data suggest that loss of CD99 may impair leukemogenesis in TET2/FLT3 but not TET2/NRAS AMLs. To determine if CD99 is required for LSC function, we performed secondary transplants of one million BM cells from primary recipients into lethally irradiated WT recipients. Mice transplanted with CD99 KO TET2/FLT3 BM, compared with those transplanted with CD99 WT TET2/FLT3 BM, demonstrated fewer donor-derived cells in the PB. While CD99 WT TET2/FLT3-transplanted mice succumbed to AML at a median of 11 weeks, all CD99 KO TET2/FLT3-transplanted mice survived beyond 16 weeks. PB smears from CD99 KO TET2/FLT3 mice demonstrated leukocytes with condensed chromatin and lobulated nuclei, indicative of restored hematopoietic maturation. Finally, in vivo O-propargyl-puromycin assays showed that loss of CD99 resulted in increased protein synthesis in LSCs (LSK CD48 +CD150 -) (fold-change=1.69±0.18, n=3 for each group). In contrast, mice secondarily transplanted with CD99 KO TET2/NRAS BM exhibited similar engraftment, survival, and morphologic evidence of AML as those transplanted with CD99 WT TET2/NRAS BM. In conclusion, our data demonstrate that LSC function in AML driven by mutated FLT3, but not mutated NRAS, is dependent on maintenance of regulated protein synthesis by CD99. We speculate that this differential dependency may reflect a different cell-of-origin, as recent work has shown that AMLs associated with FLT3-ITD exhibit particularly primitive cellular hierarchies (PMID: 35618837). Finally, our results suggest that certain high-risk AML sub-types associated with elevated CD99 expression may be highly sensitive to strategies disrupting protein synthesis to deplete LSCs.

ATP1A1/BCL2L1 predicts the response of myelomonocytic and monocytic acute myeloid leukemia to cardiac glycosides

Myelomonocytic and monocytic acute myeloid leukemia (AML) subtypes are intrinsically resistant to venetoclax-based regimens. Identifying targetable vulnerabilities would limit resistance and relapse. We previously documented the synergism of venetoclax and cardiac glycoside (CG) combination in AML. Despite preclinical evidence, the repurposing of cardiac glycosides (CGs) in cancer therapy remained unsuccessful due to a lack of predictive biomarkers. We report that the ex vivo response of AML patient blasts and the in vitro sensitivity of established cell lines to the hemi-synthetic CG UNBS1450 correlates with the ATPase Na+/K+ transporting subunit alpha 1 (ATP1A1)/BCL2 like 1 (BCL2L1) expression ratio. Publicly available AML datasets identify myelomonocytic/monocytic differentiation as the most robust prognostic feature, along with core-binding factor subunit beta (CBFB), lysine methyltransferase 2A (KMT2A) rearrangements, and missense Fms-related receptor tyrosine kinase 3 (FLT3) mutations. Mechanistically, BCL2L1 protects from cell death commitment induced by the CG-mediated stepwise triggering of ionic perturbation, protein synthesis inhibition, and MCL1 downregulation. In vivo, CGs showed an overall tolerable profile while impacting tumor growth with an effect ranging from tumor growth inhibition to regression. These findings suggest a predictive marker for CG repurposing in specific AML subtypes.

Role of Diacylglycerol Kinases in Acute Myeloid Leukemia.

Diacylglycerol kinases (DGKs) play dual roles in cell transformation and immunosurveillance. According to cancer expression databases, acute myeloid leukemia (AML) exhibits significant overexpression of multiple DGK isoforms, including DGKA, DGKD and DGKG, without a precise correlation with specific AML subtypes. In the TGCA database, high DGKA expression negatively correlates with survival, while high DGKG expression is associated with a more favorable prognosis. DGKA and DGKG also feature different patterns of co-expressed genes. Conversely, the BeatAML and TARGET databases show that high DGKH expression is correlated with shorter survival. To assess the suitability of DGKs as therapeutic targets, we treated HL-60 and HEL cells with DGK inhibitors and compared cell growth and survival with those of untransformed lymphocytes. We observed a specific sensitivity to R59022 and R59949, two poorly selective inhibitors, which promoted cytotoxicity and cell accumulation in the S phase in both cell lines. Conversely, the DGKA-specific inhibitors CU-3 and AMB639752 showed poor efficacy. These findings underscore the pivotal and isoform-specific involvement of DGKs in AML, offering a promising pathway for the identification of potential therapeutic targets. Notably, the DGKA and DGKH isoforms emerge as relevant players in AML pathogenesis, albeit DGKA inhibition alone seems insufficient to impair AML cell viability.

Genome-Based Medicine for Acute Myeloid Leukemia: Study and Targeting of Molecular Alterations and Use of Minimal Residual Disease as a Biomarker

Acute myeloid leukemia (AML) is a highly heterogeneous hematologic malignancy characterized by the clonal proliferation of hematopoietic stem and progenitor cells (HSPCs) and blockade of differentiation and proliferation of immature myeloid cells that accumulate in bone marrow at the expense of normal hematopoiesis. AMLs originate from the expansion of HSPCs progressively acquiring somatic mutations. The development of high-throughput sequencing techniques has helped to discover the genetic heterogeneity and complexity of AMLs, revise diagnostic and prognostic criteria, and to identify new therapeutic targets. These studies have allowed the identification of several recurrent driver mutations and the definition of a rational molecular classification of these tumors. In parallel, the development of techniques for the determination of single-cell mutational profiling has considerably contributed to understanding the clonal heterogeneity and evolution of AMLs. The acquisition of these genetic data coupled with the identification of molecular therapeutic targets has determined a considerable expansion of the therapeutic armamentarium, with the development of several new drugs highly active against specific AML subtypes. These developments have increased the interest and the need for sensitive techniques for the identification of minimal residual disease, the population of leukemia cells that survives despite morphological remission and causes disease relapse.

Murine Models of Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a phenotypically and genetically heterogeneous hematologic malignancy. Extensive sequencing efforts have mapped the genomic landscape of adult and pediatric AML revealing a number of biologically and prognostically relevant driver lesions. Beyond identifying recurrent genetic aberrations, it is of critical importance to fully delineate the complex mechanisms by which they contribute to the initiation and evolution of disease to ultimately facilitate the development of targeted therapies. Towards these aims, murine models of AML are indispensable research tools. The rapid evolution of genetic engineering techniques over the past 20 years has greatly advanced the use of murine models to mirror specific genetic subtypes of human AML, define cell-intrinsic and extrinsic disease mechanisms, study the interaction between co-occurring genetic lesions, and test novel therapeutic approaches. This review summarizes the mouse model systems that have been developed to recapitulate the most common genomic subtypes of AML. We will discuss the strengths and weaknesses of varying modeling strategies, highlight major discoveries emanating from these model systems, and outline future opportunities to leverage emerging technologies for mechanistic and preclinical investigations.

NKL Homeobox Genes NKX2-3 and NKX2-4 Deregulate Megakaryocytic-Erythroid Cell Differentiation in AML.

NKL homeobox genes encode transcription factors that impact normal development and hematopoietic malignancies if deregulated. Recently, we established an NKL-code that describes the physiological expression pattern of eleven NKL homeobox genes in the course of hematopoiesis, allowing evaluation of aberrantly activated NKL genes in leukemia/lymphoma. Here, we identify ectopic expression of NKL homeobox gene NKX2-4 in an erythroblastic acute myeloid leukemia (AML) cell line OCI-M2 and describe investigation of its activating factors and target genes. Comparative expression profiling data of AML cell lines revealed in OCI-M2 an aberrantly activated program for endothelial development including master factor ETV2 and the additional endothelial signature genes HEY1, IRF6, and SOX7. Corresponding siRNA-mediated knockdown experiments showed their role in activating NKX2-4 expression. Furthermore, the ETV2 locus at 19p13 was genomically amplified, possibly underlying its aberrant expression. Target gene analyses of NKX2-4 revealed activated ETV2, HEY1, and SIX5 and suppressed FLI1. Comparative expression profiling analysis of public datasets for AML patients and primary megakaryocyte–erythroid progenitor cells showed conspicuous similarities to NKX2-4 activating factors and the target genes we identified, supporting the clinical relevance of our findings and developmental disturbance by NKX2-4. Finally, identification and target gene analysis of aberrantly expressed NKX2-3 in AML patients and a megakaryoblastic AML cell line ELF-153 showed activation of FLI1, contrasting with OCI-M2. FLI1 encodes a master factor for myelopoiesis, driving megakaryocytic differentiation and suppressing erythroid differentiation, thus representing a basic developmental target of these homeo-oncogenes. Taken together, we have identified aberrantly activated NKL homeobox genes NKX2-3 and NKX2-4 in AML, deregulating genes involved in megakaryocytic and erythroid differentiation processes, and thereby contributing to the formation of specific AML subtypes.

Clinical Applications of MicroRNAs in Acute Myeloid Leukemia: A Mini-Review.

MicroRNAs (miRs) are short non-coding RNAs, typically 18-25 nucleotides in length, that are critically important, through their direct effects on target mRNAs, in a variety of cellular processes including cell differentiation, proliferation and survival. Dysregulated miR expression has been identified in numerous cancer types including acute myeloid leukemia (AML). From a clinical standpoint, several miRs have been shown to associate with prognosis in AML patients. Furthermore, they also carry the potential to be used as biomarkers and to inform medical decision making. In addition, several preclinical studies have provided strong rationale to develop novel therapeutic strategies to target miRs in AML. This review will focus on potential clinical applications of miRs in adult AML and will discuss unique miR signatures in specific AML subtypes, their role in prognostication and response to therapy, as well as miRs that are promising therapeutic targets and ongoing clinical trials directed towards targeting clinically relevant miRs in AML that could allow for improvements in current treatment strategies.

Immune Gene Expression Profiling in Children and Adults with Acute Myeloid Leukemia Identifies Distinct Phenotypic Patterns

Therapeutic strategies in patients with acute myeloid leukemia (AML) have remained relatively unchanged over the last 30 years. AML is only cured in 35-40% of patients <60 years of age and in 5-15% of patients >60 years of age. Elderly patients who are unfit to receive intensive salvage regimens can only be offered low-intensity therapy or best supportive care. In children, AML accounts for 20% of leukemias. Although intensive multi-agent chemotherapy in conjunction with improved supportive care has increased survival rates to 60-70%, 30-40% of children with AML relapse and only one-third of them will survive to adulthood. The development and delivery of new therapeutic strategies for high-risk AML, including immunotherapy, therefore remains a priority.Tumor phenotypes are dictated not only by the neoplastic cell component, but also by the tumor microenvironment (TME), which includes immune and inflammatory cells and mediators. We have previously shown that the treatment of primary AML cells with interferon (IFN)-γ in vitro induces functional indoleamine 2,3-dioxygenase-1 (IDO1), a molecule with potent immunosuppressive properties, in 50% of unselected cases (‘IFN-γ responders‘). With a median follow-up of 8 years, these IFN-γ responders experienced a significantly shorter event-free survival and overall survival.This multi-institutional study was undertaken to collect comprehensive immune profiles across the biological heterogeneity of childhood and adult AML, with the aim to implement new molecularly targeted agents for patients with specific immunologic subtypes of AML. We employed the nCounter™ system (NanoString Technologies, Seattle, USA), a medium-throughput, fully automated digital platform, to characterize bone marrow (BM) specimens from 70 patients with non-promyelocytic AML (42 children and 28 adults). Ninety BM samples (63 from patients with de novo AML, 18 from patients with AML in complete remission [CR] and 9 from patients with relapsed AML) were analyzed on the nCounter® FLEX platform, using the RNA Pan-Cancer Immune Profiling Panel. This panel detects the expression of genes for 109 cell surface markers on 24 immune cell types, 30 cancer testis antigens, and >500 immune response genes. Transcriptomic data were normalized using the nSolver™ software package (alpha version 4.0). Patient age and status (disease onset, achievement of CR and disease relapse) were selected as covariates.Hierarchical clustering identified patient subgroups with heightened expression of CD8 T-cell, T helper type 1 (Th1)-cell, B-cell and cytotoxicity-related genes, including genes related to natural killer (NK)-cell function, in the leukemia microenvironment (“immune enriched” patients) (Fig. 1A). Interestingly, a select group of patients over-expressed genes associated with monocyte/macrophage, neutrophil and mast cell functions, with low expression levels of T-cell and B-cell genes. AML patients with ‘immune enriched’ gene profiles also expressed CD8A , IFNG , FOXP3 , the T-cell chemoattractants CXCL9 , CXCL10, and inhibitory molecules including IDO1 and the immune checkpoints LAG3 , CTLA4 and PDL1 . This immune landscape is consistent with a ‘T-cell inflamed’ phenotype that might underpin the establishment of adaptive immune resistance and immune escape. Gene set analyses identified differences in immune gene expression levels between childhood and adult AMLs, as well as patients with newly diagnosed AML, relapsed AML or AML in CR (Fig. 1B-C). In particular, genes highly expressed in adult AMLs compared with childhood disease included chemokine, dendritic cell and macrophage genes . In contrast, immune genes down-regulated in adult compared with childhood AMLs included integrin family members, cytokine receptors, T-cell receptor complex components (CD3EAP ) and lipocalin-2 (LCN2 ), a neutrophil gelatinase-associated molecule implicated in suppression of tumor invasiveness and metastasis.In conclusion, our analysis has identified heterogeneous immunological profiles in children and adults with AML at different disease stages. From a clinical standpoint, ‘immune enriched’ AMLs might be amenable to immunotherapy approaches tailored to the BM microenvironment, including blockade of co-inhibitory molecules and/or small-molecule IDO1 inhibitors.Grant support: Roger Counter Foundation, UK; Qatar National Research Fund (#NPRP8-2297-3-494). [Display omitted] DisclosuresWarren:NanoString Technologies: Employment, Equity Ownership. Hood:NanoString Technologies: Employment, Equity Ownership. Danaher:NanoString Technologies: Employment, Equity Ownership. Cesano:NanoString Technologies: Employment, Equity Ownership. Beechem:NanoString Technologies: Employment, Equity Ownership. Tasian:Gilead Sciences, Inc.: Research Funding; Incyte Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Rutella:NanoString Technologies, Seattle, USA: Research Funding.

Setbp1 Overexpression Acts in the Place of Class-Defining Somatic Mutations to Drive Mouse and Human FLT3-ITD-Mutant AMLs

Setbp1 overexpression acts in the place of class-defining somatic mutations to drive mouse and human FLT3-ITD-mutant AMLs Suruchi Pacharne,1,2 Oliver M. Dovey,1 Jonathan L. Cooper,1 Muxin Gu,1,2 MS Vijaybaskar,1,2 Mathias J. Friedrich,1,5 Malgorzata Gozdecka,1,2 Sandeep S. Rajan,1,4, Etienne De Braekeleer,1,2 Maxim Barenboim,5,6 Grace Collord,1,2 Hannes Ponstingl,1 Ruben Bautista,1 Milena Mazan,1,8 Roland Rad,5,6 Konstantinos Tzelepis,1,7 Penny Wright,3 and George S. Vassiliou1,2,9* Abstract Internal tandem duplications in FLT3 (FLT3-ITD) are found in 30% of acute myeloid leukemia (AML) cases and impart a poor prognosis. FLT3-ITD commonly synergizes with class-defining mutations such as chimeric fusion genes or mutations in NPM1, RUNX1, CEBPA or MLL to drive AML. However, 20% of FLT3-ITD-mutant AMLs bare no class-defining mutations and the mechanisms of acute leukemic transformation in these cases are unknown. To identify pathways that can drive FLT3-ITD-mutant AML in the absence of class-defining mutations, we performed an insertional mutagenesis (IM) screen in Flt3-ITD mice using the Sleeping Beauty transposon system, activated by the Mx1-Cre recombinase in hematopoietic stem cells. All mice developed acute leukemia, predominantly AML, after a median latency of 73 days (Figure A). Analysis of transposon insertions in 38 Flt3-ITD/IM leukemias identified common integration sites (CISs) in 22 loci (Figure B). The most commonly "hit" genes were Setbp1 (20/38), Ets1 (11/38), Ash1l (8/38), Notch1 (8/38), Erg (7/38), Flt3 (6/38) and Runx1 (5/38) (Figure B). Of these, Setbp1 and Runx1 were unique to Flt3-ITD and not identified as CISs in insertional mutagenesis screens of wild type, Npm1c or BCR-ABL-expressing mice. Transposon insertions in Setbp1, primarily located upstream of its first coding exon, were associated with Setbp1 and Hoxa mRNA overexpression and were invariably associated with AML development (Figure B). These findings propose that overexpression of wild type SETBP1 may collaborate with FLT3-ITD to drive leukemogenesis in human AMLs lacking mutations known to collaborate with mutant FLT3. Corroborating this, we found that SETBP1 expression was higher in human FLT3-ITD-mutant AMLs lacking class-defining mutations and in those with RUNX1 mutations (Figure C). We go on to show that Setbp1 insertions activate a Hoxa gene signature such that shares significant similarities, but also specific differences to those driven by mutant Npm1 and MLL fusion genes. We go on to show, using CRISPR-gRNA, that whilst Flt3ITD/+/SETBP1IM+AMLs are entirely dependent on Setbp1 expression, Flt3ITD/+/Npm1cA/+AMLs are not, but do depend on the expression of the homebox gene Nkx2.3. Our findings propose that SETBP1 overexpression activates a gene expression pattern that collaborates with FLT3-ITD to drive many human AMLs and that this combination represents a specific subtype of AML amongst AMLs lacking class-defining mutations. To identify genetic vulnerabilities of this AML subtype, we performed genome-wide CRISPR-Cas9 recessive screens in primary murine Flt3ITD/+SETBP1IM+AMLs and identified more than 2000 genetic vulnerabilities, of which 677 were not required for the survival of HPC7 non-leukemic hematopoietic cells including &amp;gt;100 "druggable" genes such as Brd3, Ezh2 and Hmgcr (Figure D). Collectively our study: i) identifies SETBP1overexpression as a non-genetic alteration driving a subgroup of FLT3-ITD mutant AMLs lacking class-defining somatic mutations and ii) goes on to define the genetic vulnerabilities of such AMLs as a starting point for the development of targeted therapies. Figure Disclosures Vassiliou: Kymab Ltd - Monoclonal antibody company. Currently not working in myeloid cancers or clonal haematopoiesis.: Consultancy.

Metabolomics analysis identifies lysine and taurine as candidate prognostic biomarkers for AML-M2 patients.

There is an ongoing search for potential biomarkers for acute myeloid leukemia (AML) patients using metabolic analysis. However, only few studies to date have focused on bone marrow samples or a specific subtype of AML. In the present study, we used gas chromatography time-of-flight mass spectrometry of plasma and bone marrow supernatants to compare the metabolic characteristics of patients with AML with maturation (AML-M2). This approach identified significantly altered metabolites. We next performed pathway analysis and determined relative mRNA expression by qRT-PCR. Our results show that lysine, methionine and serine were significantly decreased in AML-M2 patients compared with healthy control. Moreover, plasma abundance of lysine was negatively associated with patients' risk stratification. Taurine had higher plasma abundance in AML-M2 patients and plasma level of taurine was positively related with AML-M2 risk status, while the expression level of taurine transporter showed a negative correlation. Receiver operating characteristic curve analysis showed these four metabolites had high diagnostic value with lysine showing the highest sensitivity and specificity. These results suggest that plasma abundances of lysine and taurine may serve as potential metabolic biomarkers for the prognosis of patients with AML-M2.

Genetic Characteristics and Outcomes By Mutation Status in a Phase 3 Study of CPX-351 Versus 7+3 in Older Adults with Newly Diagnosed, High-Risk/Secondary Acute Myeloid Leukemia (AML)

Lindsley: Jazz Pharmaceuticals: Research Funding; Takeda Pharmaceuticals: Consultancy; Medlmmune: Research Funding. Stone:AbbVie, Actinium, Agios, Argenx, Arog, Astellas, AstraZeneca, Biolinerx, Celgene, Cornerstone Biopharma, Fujifilm, Jazz Pharmaceuticals, Amgen, Ono, Orsenix, Otsuka, Merck, Novartis, Pfizer, Sumitomo, Trovagene: Consultancy; Argenx, Celgene, Takeda Oncology: Other: Data and Safety Monitoring Board/Committee: ; Novartis, Agios, Arog: Research Funding. Cortes:Astellas Pharma: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; BiolineRx: Consultancy; Sun Pharma: Research Funding; Jazz Pharmaceuticals: Consultancy, Research Funding; Merus: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Research Funding; Immunogen: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Biopath Holdings: Consultancy, Honoraria; Forma Therapeutics: Consultancy, Honoraria, Research Funding; Daiichi Sankyo: Consultancy, Honoraria, Research Funding. Uy:GlycoMimetics: Consultancy; Astellas: Consultancy; Curis: Consultancy; Pfizer: Consultancy. Lin:Pfizer: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Honoraria. Ritchie:Celgene, Incyte, Novartis, Pfizer: Consultancy; Ariad, Celgene, Incyte, Novartis: Speakers Bureau; AStella, Bristol-Myers Squibb, Novartis, NS Pharma, Pfizer: Research Funding; Celgene, Novartis: Other: travel support; Jazz Pharmaceuticals: Research Funding; Genentech: Other: Advisory board; Celgene: Other: Advisory board; Pfizer: Other: Advisory board, travel support; agios: Other: Advisory board; Tolero: Other: Advisory board. Prebet:novartis: Honoraria; pfizer: Honoraria; pfizer: Honoraria; novartis: Honoraria; Boehringer Ingelheim: Research Funding. Ryan:Jazz Pharmaceuticals: Employment, Equity Ownership. Faderl:Jazz Pharmaceutics: Employment, Equity Ownership. Lancet:Pfizer: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Other: fees for non-CME/CE services ; Agios, Biopath, Biosight, Boehringer Inglheim, Celator, Celgene, Janssen, Jazz Pharmaceuticals, Karyopharm, Novartis: Consultancy.

BCL2 overexpression: clinical implication and biological insights in acute myeloid leukemia

BackgroundBCL2 protein inhibitor venetoclax (ABT-199) has been authorized by Food and Drug Administration for relapsed/refractory chronic lymphoid leukemia with 17p deletion. Although venetoclax/ABT-199 also caused cell death in acute myeloid leukemia (AML), whether it could be applied to clinical treatment needs further studies. Here, we revealed clinical implication of BCL2 overexpression in de novo adult AML, and may provide theoretical basis for targeted therapy using venetoclax.MethodsBCL2 expression was analyzed in adult AML patients from public datasets The Cancer Genome Atlas (TCGA) and confirmed by another independent cohort from our own data.ResultsBCL2 expression showed up-regulated in AML patients among TCGA data and confirmed by our own data. BCL2 overexpression was correlated with FAB-M0/M1, whereas BCL2 under-expression was related to FAB-M5. However, BCL2 expression has no effect on overall survival (OS) and leukemia-free survival (LFS) of AML patients (determined in BCL2low and BCL2high groups). Interestingly, in the BCL2low group, patients undergoing autologous or allogeneic hematopoietic stem cell transplantation (auto/allo-HSCT) had significantly better OS and LFS compared with patients only received chemotherapy, whereas, no significant difference was found in OS and LFS between chemotherapy and auto/allo-HSCT patients in the BCL2high group. BCL2 expression was found positively correlated with HOX family gene, and negatively correlated with tumor suppressor microRNA such as miR-195, miR-497, and miR-193b.ConclusionsBCL2 overexpression identified specific FAB subtypes of AML, but it did not affect prognosis. Patients with BCL2 overexpression did not benefit from auto/allo-HSCT among whole-cohort-AML and cytogenetically normal AML.

Synergistic Anti-Leukemic Activity of PARP Inhibition Combined with IMGN632, an Anti-CD123 Antibody-Drug Conjugate in Acute Myeloid Leukemia Models

BackgroundCD123 (IL-3 receptor alpha-chain) is a therapeutic target for hematological malignancies based on high expression levels in acute myeloid leukemia (AML), blastic plasmacytoid dendritic cell neoplasm (BPDCN), and other cancers. The anti-CD123 antibody-drug conjugate (ADC), IMGN632, comprises a humanized monoclonal antibody covalently linked to a DNA - alkylating cytotoxic payload which is currently in phase 1 evaluation for relapsed/refractory CD123-positive hematological malignancies (NCT03386513). Novel approaches to enhance the efficacy of ADCs are of significant therapeutic interest. Our laboratory has previously demonstrated that the Poly ADP Ribose (PARP) inhibitor, olaparib, synergistically enhances the activity of the CD33-targeted ADC, IMGN779, in preclinical AML models (Portwood S et al, ASH 2016). Based on the hypothesis that PARP inhibition will synergize with DNA damaging mechanism of IMGN632, we investigated the ability of olaparib and other PARP inhibitors (PARPi) in clinical development (talazoparib, niraparib, rucaparib, and veliparib) to enhance the therapeutic efficacy of IMGN632 across diverse human AML cell lines and primary relapsed/refractory AML samples.Materials and MethodsCD123 expression on human AML cell lines (HEL, HL60, MV411, Molm13, EOL-1, THP-1, and Kasumi-1) was quantified by flow cytometry using QuantriBrite beads. AML cells were continuously cultured for 72-96 hours with varying doses of IMGN632 (range 100pM - 100nM) and specific PARP inhibitors (range 100pM -15μM) alone and in combination. Cell viability was measured using a WST-8 colorimetric assay. Primary clinically annotated CD123+ AML cells from patients with relapsed/refractory disease were obtained under IRB-approved protocols from the Roswell Park Hematologic Procurement Shared Resource and cultured short-term in the presence of multiple cytokines plus IMGN632 +/- PARP inhibitors. Apoptosis (Annexin V/PI), cell cycle, and DNA damage (H2AX) were evaluated by flow cytometry. Additive vs. synergistic effects were determined by combination indices using Compusyn software. PARP trapping was evaluated by Western blot analysis in nuclear lysates obtained from IMGN632 +/- PARP inhibitors treated AML cells.ResultsHigh expression levels of CD123 (range 937 - 2231 CD123 molecules/cell) were detected on multiple human AML cell lines (HEL-luc, MV411, Molm13, EOL-1, and THP-1) relative to unstained negative controls. Western blot analysis of nuclear lysates from AML cells demonstrated that all PARP inhibitors had varying degrees of PARP trapping on DNA. Continuous single agent 5-day treatment with all tested PARP inhibitors resulted in dose dependent in vitro inhibition of AML cell line growth with IC50 values ranging from 360 nM (talazoparib, most potent) to 78uM (veliparib, least potent). Combination therapy using PARP inhibitors (doses ranging from 300nM - 15uM) and IMGN632 (10nM) consistently resulted in enhanced anti-leukemic effects over monotherapy (Figure 1 for example). Synergistic anti-proliferative effects were obtained across all tested AML cell lines (n=5) with combination indexes ranging from 0.3-0.7 by Compusyn analysis. Combination therapy correlated with enhanced DNA damage, tumor cell apoptosis, and cell cycle arrest of AML cells. Moreover, IMGN632 and PARPi (olaparib or talazoparib) resulted in single agent activity against clinically annotated primary relapsed/refractory AML patient samples with evidence of synergistic effects when combined in vitro.ConclusionsAddition of PARP inhibitors to IMGN632, a novel anti-CD123 antibody-drug conjugate, further enhances DNA damage effects and consistently results in synergistic in vitro anti-leukemic effects across multiple CD123+ AML cell lines and primary AML patient samples. Further studies investigating this novel combinatorial approach in specific molecular subtypes of AML with variable baseline sensitivities to PARPi are currently ongoing. Our results strongly support future investigation of PARPi as a novel class of agents with the potential to significantly enhance the efficacy of DNA-alkylating ADCs and/or cytotoxic chemotherapy for hematological malignancies. [Display omitted] DisclosuresSloss:ImmunoGen: Employment. Watkins:ImmunoGen Inc.: Employment. Kovtun:ImmunoGen Inc.: Employment. Adams:ImmunoGen Inc.: Employment. Wang:Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Speakers Bureau; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy; Novartis: Speakers Bureau; Jazz: Speakers Bureau; Jazz: Speakers Bureau; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees.

NSAID consumption and risk of acute myeloid leukemia: a national population-based case-control study.

BackgroundMost cases of acute leukemia arise without identifiable risk factors. Studies investigating the impact of autoimmune diseases and infections on leukemogenesis have revealed conflicting results. If inflammation increases the risk of acute myeloid leukemia (AML), nonsteroidal anti-inflammatory drug (NSAID) use may decrease the risk of leukemia.MethodsWe conducted a case-control study of 3,053 patients with AML diagnosed between 2000 and 2013, who were registered in the Danish National Acute Leukemia Registry, and 30,530 population controls matched on sex and age. We identified prescriptions through the Danish National Health Service Prescription Database. We used conditional logistic regression analysis to compute ORs associating AML with NSAID use overall, in patients with inflammatory diseases, and for specific AML subtypes (de novo AML, AML related to previous hematological disease, ie, secondary AML [sAML], or therapy-related AML [tAML; exposed to previous cytotoxic therapy]).ResultsOverall, NSAID use was not associated with a lower risk of AML (OR 1.1, 95% CI=1.0–1.2), de novo AML (OR 1.0, 95% CI=0.9–1.1), and sAML/tAML (OR 1.3, 95% CI=1.1–1.5). In addition, in patients with known inflammatory diseases, NSAIDs did not affect AML risk (OR 0.9, 95% CI=0.5–1.6). Number of prescriptions, type of NSAID, age, or sex did not influence the results.ConclusionIn line with our recent findings that showed no association between autoimmune diseases and infections and de novo AML, NSAID use was not found to reduce the risk of AML.