Hallmark Of Acute Myeloid Leukemia Research Articles

Cohesin RAD21 Gene Promoter Methylation in Patients with Acute Myeloid Leukemia.

Aberrant gene promoter methylation is one of the hallmarks of Acute Myeloid Leukemia (AML). RAD21 is an important gene, implicated in sister chromatids cohesion, DNA repair, the regulation of gene transcription, apoptosis and hematopoiesis. In this study, we investigate the possible implication of RAD21 promoter methylation in AML pathogenesis using a cohort of AML patients and a cohort of healthy individuals. RAD21 promoter methylation was found in 24% of patients and in none of the controls (p = 0.023), indicating a possible contribution to AML development. Interestingly, a statistically higher frequency of RAD21 methylation was observed in patients with trisomy 8 (9/21, 42.9%, p = 0.021), while none of the patients with aberrations of chromosome 11 had RAD21 gene promoter methylation (0%, 0/11, p = 0.048). Patients with monosomal and complex karyotypes showed low frequencies of RAD21 methylation (7.7% and 15.4%, respectively) without reaching statistical significance. Moreover, ASXL1 mutations were not found to be associated with RAD21 methylation. This is the first study which provides evidence for a possible pathogenetic role of RAD21 promoter methylation in AML development and especially in AML with trisomy 8. Further studies of RAD21 promoter methylation in large series of different AML genetic subgroups may contribute to the elucidation of AML pathogenesis and to the identification of new epigenetic biomarkers with diagnostic and prognostic value.

Abstract 5628: Aberrant 3'UTR processing regulates a tumor suppressor network in AML

Abstract Differentiation blockade is a hallmark of acute myeloid leukemia (AML). While inactivating mutations in tumor suppressor genes (TSGs) have been well-studied, non-genetic mechanisms that impair TSGs to promote myeloid differentiation and influence drug response remain poorly understood. In this study, we performed genome-wide loss-of-function CRISPR/Cas9 screens to identify suppressors of myeloid differentiation. These screens converged on the zinc finger transcriptional repressor, ZBTB7A whose ablation restricted myeloid maturation and promoted a dysregulated inflammatory state to protect leukemic cells from undergoing differentiation. Moreover, genetic deletion of ZBTB7A or its paralog, ZBTB7B impeded leukemia differentiation and conferred resistance to differentiation-inducing agents. Mechanistically, we found that leukemia cells hijack 3’UTR processes to reduce mRNA expression of the tumor suppressor gene paralogs ZBTB7A and ZBTB7B. These findings provide a molecular framework of altered 3’UTR regulation that orchestrate a tumor suppressive function and promote resistance to AML differentiation-based therapies. Citation Format: Eric Wang, Cuijuan Han, Alexander Calderon, Abimbo Lawal, Sakthi Harini Rajendran, Sadik Karma, Nidhi Hariramani, Iannis Aifantis, Aristotelis Tsirigos. Aberrant 3'UTR processing regulates a tumor suppressor network in AML [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 5628.

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.

WITHDRAWN: MiR-3913-5p is a novel key regulator of differentiation arrest in acute myeloid leukemia

BackgroundMicroRNAs (miRNAs) are a class of small non-coding RNAs that have been shown to play a crucial role in normal hematopoiesis and leukemogenesis. Differentiation arrest is the hallmark of acute myeloid leukemia (AML). However, key miRNAs regulating differentiation arrest in AML remain unclear. MethodsSmall RNA-seq was performed in paired bone marrow samples at initial diagnosis vs. complete remission (CR) and differentially expressed miRNAs (DEMs) were analyzed for each patient. The mRNA levels of significantly upregulated miRNAs in all the patients were assayed during myeloid differentiation induced by all-trans retinoic acid (ATRA) in HL-60 and NB4 cells by Q-PCR. HL-60 and NB4 cells were infected with miR-3913-5p lentivirus (Lenti-miR-3913-5p), and the myeloid differentiation rate was assessed by flow cytometry as well as May–Grünwald Giemsa staining. CCK-8 assay was performed to detect the proliferation of cells. ResultsmiR-3913-5p and other 17 miRNAs were significantly upregulated at initial diagnosis vs. complete remission, and decreased in ATRA-induced myeloid differentiation of HL-60 cells and NB4 cells. Overexpression of miR-3913-5p inhibited myeloid differentiation, while had little effect on proliferation, in HL-60 and NB4 cells. ConclusionsmiR-3913-5p is a novel key regulator of cell differentiation arrest in AML.

Aberrant 3'UTR Processing Regulates Leukemogenesis and Therapy Resistance

A block in cellular differentiation is a hallmark of acute myeloid leukemia (AML) that promote clonal expansion and influence therapy response 1,2. It has become evidently clear that non-genetic mechanisms are critical to prevent malignant cells to resume its lineage maturation trajectory 3,4. However, there is still an incomplete understanding of gene regulatory pathways that induce differentiation arrest of leukemia cells. Here, we applied an immunophenotypic readout and positive selection CRISPR/Cas9 screens to identify suppressors of leukemia differentiation. These screens converged on ZBTB7A, a zinc finger transcription factor whose loss-of-function restricted AML maturation in the prescence of differentiation-inducing agents. Mechanstically, ZBTB7A ablation led to an elevated inflammatory state to stimulate leukemia proliferation. In addition, we found that genetic deletion of ZBTB7A and its paralog, ZBTB7B cooperatively suppresses myeloid differentiation and promotes resistance to several clinical compounds. Lastly, we identify that leukemic cells hijack multiple upstream 3'UTR processes, such as alternative polyadenylation and mRNA decay to downregulate ZBTB7A and ZBTB7B expression. Together, these data provide fundamental insight into non-genetic mechanisms that impede myeloid differentiation and mediate drug resistance.

Iadademstat and Gilteritinib for the Treatment of FLT3-Mutated Relapsed/Refractory Acute Myeloid Leukemia: The Frida Study

Background: Frontline and relapsed /refractory (R/R) acute myeloid leukemia (AML) patients carrying fms-related tyrosine kinase 3 [FLT3]-mutations (FLT3 mut+) benefit from FLT3 inhibitors (FLT3i) that have emerged as active therapeutic agents for this subset of AML encompassing up to 30-40% of all AML cases. The FLT3i gilteritinib, as monotherapy for R/R AML pts, has improved outcomes but the duration of remission achieved is transient and often brief. The Phase 3 randomized ADMIRAL trial of gilteritinib demonstrated a 20% CR rate and 2.8 months event-free-survival (EFS) (Perl, et al., NEJM 2019). The development of new targeted agents which synergize with FLT3 inhibitors, and ideally also inhibit the emergence of resistance, is therefore a major medical need in AML. Iadademstat (iada/ORY-1001) is a specific, oral, potent, covalent inhibitor of the epigenetic Lysine-Specific Demethylase 1 (LSD1/KDMA1) enzyme. Epigenetic dysregulation is a hallmark of AML. Up to 70% of recurring mutations in AML patients target epigenetic regulators of gene expression, resulting in myeloid differentiation blockade and enhanced leukemic stem cell renewal, underscoring the potential of epigenetic therapies to change the natural history of the disease. Preclinically, iada produces striking synergy with FLT3is, particularly gilteritinib in FLT3 wild-type and FLT3 mut+ AML cells and in derived cell lines resistant to venetoclax, azacitidine and other FLT3is (Sacilotto et al., 2022 Eur J. of Cancer 174S1). The novel MOAs generating this synergy in combinations include activation of a pro-differentiating epigenetic transcriptional program with simultaneous suppression of MYC-driven target genes (Yashar, et al. 2023, Mol. Cancer Res). The recently completed Phase 2 study of iada in combination with azacitidine (ALICE) produced a high rate of composite remission, including complete remission (CR) and CR with incomplete count recovery (CRi) (mostly MRD negative), as well as durable responses, in treatment naïve, unfit AML patients (pts), without exacerbating the toxicity profile of azacitidine (Salamero et al., 2022, Blood S1). The FRIDA study (NCT05546580) aims to establish the safety, tolerability, and the recommended phase 2 dose (RP2D) of the combination of iada plus gilteritinib in FLT3 mut+ R/R AML. Methods: Adult pts with body weight ≥50 Kg and ECOG 0-2, with FLT3 mut+ R/R AML, after no more than 2 prior lines of therapy, are enrolling. Certain patients with prior exposure to FLT3is including gilteritinib may be eligible. Up to 18 pts, in a 3+3 escalation phase, will receive iada at doses of 75 to 150 ug, orally, in 5 days ON - 2 days OFF schedule, with continuous gilteritinib, at 120 mg/day orally. In the expansion phase, up to 14 pts at the selected safe and pharmacologically active dose/s (determined based on all available data from escalation pts including PK, target engagement (TE), safety, tolerability, and emerging activity) will be enrolled. Primary endpoints of the study are safety and RP2D determination, based on the same 5 criteria outlined above. Bayesian posterior probability efficacy monitoring will be performed periodically for each dose cohort in the expansion phase. Bayesian efficacy futility and early stopping boundary will be applied during the monitoring. Posterior probability criterion (Prob (CR>0.3) ≥ 0.60) at the end of the study will warrant additional development. The safety of the combination treatment will also be continuously evaluated during expansion following a Bayesian design stopping rule.Secondary endpoints include overall survival, EFS, CR, CR/CR with partial hematologic recovery (CRh), overall response rates, time to response, duration of response, and transfusion rate. Exploratory endpoints include measurable residual disease and gene mutational analysis. At the time of the submission, FRIDA is enrolling a second dose level cohort in escalation phase and plans to have the 15 sites open to accrual in the US by the end of 2023. Additional sites will be added for a subsequent randomized controlled double-blinded FRIDA 2 study to assess the efficacy of the iada and gilteritinib combination in R/R FLT3 mut+ AML.

Self-Supporting Human Acute Myeloid Leukemia (AML) Bone Marrow Organoids Established from Patient-Derived Xenografts Mimic the Human AML Microenvironment, with Distinct Cytokine Kinetics Correlating with Dynamic Cell Proliferation and Metabolic Shifts

Hallmarks of acute myeloid leukemia (AML) include (1) formation of a self-supporting and self-regulating tumor microenvironment (TME) in the bone marrow (BM) rendering pathological niches that orchestrate retention, proliferation and survival of AML blasts, and (2) a dynamic metabolism that promotes therapy resistance and disease progression. Efforts to understand AML biology and enhance current therapies are hampered by the paucity of state-of-the-art human models that can recreate the three-dimensional (3D) BM TME and recapitulate the heterogeneity, complexity, dynamics, and metabolic shifts observed in human disease. Previously, we described the development of the first long-term self-propagating serum- and cytokine-free human BM organoid generated from an AML patient-derived xenograft (PDX) that captured metabolic shifts during AML progression. Here, we characterize the dynamic changes observed in the self-constituted TME with respect to AML proliferation, metabolism, and upon reconstitution of secondary organoids. BM was collected from NSG mice engrafted with human primary AML cells (PDX-AML model) or naïve control mice and seeded into polyurethane scaffolds in serum- and cytokine-free medium at 4e6 cells per scaffold, optimized previously. Scaffolds were recharged with autologous PDX-AML BM on day (D) 13 and cultured until D70. Organoids were assessed for (1) inter- and intra-scaffold AML kinetics, (2) the TME, (3) metabolic composition and (4) propagation into secondary organoids. AML cell kinetics from organoid culture supernatants reflected those of scaffold-extracted populations (n=3). CD33 +CD44 + AML cells expanded and/or were maintained from 7% (D0) to >10% (D70), being >15% at most culture timepoints. Confocal microscopy revealed reconstitution of typical AML-TME niche interactions within organoids, including expression of fibronectin, VCAM-1 and N-Cadherin, and formation of proliferative niches composed of AML cells with heterogeneous Osteopontin- and Osterix-expressing support cells. Human AML-supportive cytokines were consistently observed in organoid supernatants (N=3), with SDF-1, IL-1α, IL-8, GM-CSF, FGF-2 and Osteopontin present throughout the culture; some cytokines had biphasic kinetics with factors critical for TME formation present early (e.g. Osteopontin, GM-CSF), and those required for AML proliferation dominant later (e.g. FLT3L, IL-8), coinciding also with metabolic shifts during culture. Despite the absence of IL-6, single molecule RNA fluorescence in situ hybridization confirmed IL-6 expression by niche-resident AML blasts suggesting autocrine signaling networks beyond those identified in supernatants. Cells from D70 organoids were able to re-establish secondary organoids (n=2) which also exhibited long-term (D70) AML maintenance (>11% blasts), and regenerated the AML TME. Previously, we identified 3 culture stages defined by metabolic shifts: 1) pre/early recharge (D4-D19), (2) intermediate (D25-D40) and, (3) late (from D55) where organoids exhibited highest proliferation. To further characterize these shifts, intracellular metabolites were extracted from organoids (n=5) at D10, D25, D40, D55 and D70, analyzed with gas-chromatography mass spectrometry (GC-MS) and evaluated through unsupervised hierarchical clustering and multivariate analysis. Significance Analysis of Microarrays (SAM) analysis demonstrated dysregulation of alanine, serine, isoleucine, glycolate, hexanoic acid and glycolysis when comparing stage 1 with stage 2, and in isoleucine, leucine, serine, succinate and tryptophan metabolism when comparing stage 2 with stage 3. When proliferative and non-proliferative timepoints were compared, amino acid and glycolysis-driven metabolic switches were identified, typical of AML metabolism. We have characterized a long-term 3D human AML BM biomimicry in serum- and cytokine-free conditions with a self-organizing TME that recapitulates many features of AML BM, including robust human AML cell maintenance within niches, cytokine and metabolic kinetics to support different phases of progression, and the ability to propagate into secondary organoids. This dynamic, physiologically-relevant ex vivo platform can enable studies in human AML pathogenesis/progression and is poised to test novel therapeutics directed towards AML cells, the TME and metabolic targets.

SUMO E3 Ligase ZMIZ1 Drives Leukemogenesis Via Transcriptional Deregulation in Acute Myeloid Leukemia

Transcriptional deregulation is one of the hallmarks of acute myeloid leukemia (AML), which causes the aberrant expression of genes essential for hematopoiesis, proliferation, stemness et, al. Emerging evidence has demonstrated that transcription is regulated by post-translational modification of transcription factors, such as SUMOylation, and the recruitment of co-regulators, such as epigenetic regulators. Here, we conducted a CRISPR-Cas9 screen using sgRNAs targeting epigenetic regulators and a SUMO E3 ligase to explore essential regulators for hematopoiesis and leukemogenesis. Intriguingly, sgRNAs targeting ZMIZ1 was identified to be significantly decreased in proliferated leukemia cells and enriched in differentiated ones. ZMIZ1 (also known as ZIMP10 or RAI17) is a SUMO E3 ligase and transcriptional co-regulator of the Protein Inhibitor of Activated STAT (PIAS)-like family. Moreover, we found ZMIZ1 was highly expressed in AML and related to unfavorable outcomes, suggesting a potential diagnostic and prognostic target for AML. Next, we analyzed the function of ZMIZ1 in AML using both in vivo and in vitro approaches. We knocked down the expression of ZMIZ1 in MOLM-13, THP1, OCI-AML3, HL60, and NB4 cells and found that knockdown of ZMIZ1 suppressed cell viability and induced apoptosis in all five cell lines. Meanwhile, knockdown of ZMIZ1 enhanced the sensitivity to chemotherapy (such as Ara-C). Additionally, knockdown of ZMIZ1 induced cell differentiation in NB4 cells. Moreover, we established conditional knockout mouse models (Zmiz1 flox/floxMx1-Cre), in which Zmiz1 was deleted with the Mx1-Cre transgene. We isolated the bone marrow of these mice and transfected it with the MLL-AF9 fusion gene, followed by transplantation into recipient mice. The conditional knockout was induced after two weeks of transfection. We found that inactivation of Zmiz1 significantly prolonged survival and relieved the leukemic burden. To examine the regulatory function of ZMIZ1 in AML, we conducted ChIP-seq analysis in MOLM-13, THP1, OCI-AML3, HL60, and NB4 cells using antibodies against ZMIZ1. The binding peaks of ZMIZ1 differ in AML cell lines, as fewer than 30% of binding sites were overlapped between different cells. However, the binding motifs for ZMIZ1 showed a similar pattern. Motifs bound by MYB, CENPE, and RUNX1 were enriched in all five cell lines. To test the regulatory effects of ZMIZ1 on these transcription factors, we conducted luciferase reporter assays and found that ZMIZ1 enhanced the transcriptional activity of MYB. Taken together, our study suggests an essential function of ZMIZ1 in leukemogenesis, presumably via SUMOylating essential transcription factors, and sheds new light on AML targeting therapy probably by targeting ZMIZ1.

Co‐Targeting c‐Myc and Bcl‐2 by Oral Small Molecule Combination of WBC100 and Venetoclax Effectively Controls Acute Myeloid Leukemia in Preclinical Models

AbstractUncontrolled proliferation and apoptosis evasion are two hallmarks of acute myeloid leukemia (AML), but the molecular mechanisms remain poorly understood. In this study, it is demonstrated that the double over‐expresser of oncoprotein c‐Myc and anti‐apoptotic protein Bcl‐2 is a critical “genetic overdrive” of proliferation and apoptosis evasion in AML and is associated with poor genetic alterations. Double‐knockdown of c‐Myc/ Bcl‐2 synergistically kills AML cells in vitro and in vivo. Moreover, a novel oral small molecule combination co‐targeting c‐Myc and Bcl‐2 with WBC100 and Venetoclax (VEN) at low doses are developed. Importantly, the study shows that this combination results in deep and durable remissions of AML, and its efficacy is superior to the frontline combination of Venetoclax and hypomethylation azacitidine (AZA) in AML mouse models and PDX models from relapsed or refractory AML patients. Mechanically, Bcl‐2 knockdown induces mitochondrial outer membrane permeabilization and c‐Myc‐knockdown impairs mitochondria biogenesis. Co‐targeting c‐Myc/Bcl‐2 reciprocally abrogates over‐proliferation and apoptosis resistance via forming a double hit to mitochondrial biogenesis and apoptosis machinery. The findings for the first time demonstrate that co‐targeting c‐Myc/Bcl‐2 by the novel oral small molecule combination of WBC100/Venetoclax is a promising and convenient therapy for AML and support its clinical trial.

Iadademstat in combination with gilteritinib for patients with mutated FLT3 relapsed/refractory acute myeloid leukemia.

TPS7075 Background: Epigenetic dysregulation is a hallmark of acute myeloid leukemia (AML) and is caused by recurrent translocations and/or mutations in chromatin regulators and transcription factors, resulting in myeloid differentiation blockade and leukemic stem cell renewal. Accordingly, about 70% of recurring mutations in AML patients (pts) target regulators of gene expression, underscoring the potential of epigenetic therapies to change the disease natural history. Iadademstat (iada/ORY-1001) is a specific, oral, potent, covalent inhibitor of the epigenetic Lysine-Specific Demethylase 1 (LSD1/KDMA1) enzyme. In preclinical and clinical studies iada decreased leukemic stem cell survival and induced macrophage/monocytic differentiation of blasts. ALICE, a Ph2 study of iada in combination with azacitidine (aza), showed high complete remission (CR/CRi) rates and durable responses in treatment naïve, unfit AML pts without exacerbating the toxicity profile of aza (Salamero et al., Oral at ASH 2022). Despite improvements in AML therapy, relapsed and refractory (R/R) cases are frequent and contribute to the death of more than 50% of pts, particularly in those subpopulations with higher risk genetics, the largest of which (up to 30-40% AML pts) harbors fms-like tyrosine kinase 3 mutations (FLT3mut+). Use of the FLT3 inhibitor (FLT3i) gilteritinib as monotherapy for R/R pts resulted in improved outcomes but the duration of remission achieved is transient and often brief (CR rate: 20%; EFS: 2.8 months) per the ADMIRAL Ph3 study (Perl, et al., NEJM 2019). Preclinically, iada has marked synergy with FLT3i, particularly gilteritinib, in FLT3 wild-type and FLT3 mut+ AML cells and in derived cell lines resistant to venetoclax, azacitidine and FLT3is. Methods: Adult pts with ECOG 0-2 and FLT3 mut+ R/R AML, after 1 or 2 prior lines of therapy, will be enrolled. In escalation (3+3), up to 18 pts will receive iada at 75 to 150 ug, orally, in 5 days ON-2 days OFF schedule, with gilteritinib SoC. Up to approximate 14 pts will be expanded from pharmacologically active dose/s (per Project OPTIMUS). Primary endpoints of the study are safety and RP2D determination. Bayesian posterior probability efficacy monitoring will be performed periodically for each dose cohort. Bayesian efficacy futility and early stopping boundary will be applied. Posterior probability criterion (Prob (CR > 0.3) ≥ 0.60) at the end of the study will warrant further research. Safety in expansion will be evaluated continuously with a Bayesian stopping rule. Secondary endpoints include OS, EFS, CR, CR/CRh, ORR, TTR, DoR, and transfusion rate. Exploratory endpoints include PK, PD, MRD and gene mutational analysis. The study will run in 15 US sites. Additional sites will be added for a subsequent Phase 2 randomized controlled double-blinded FRIDA 2 study to assess the efficacy of this combination in R/R FLT3 mut+ AML. Clinical trial information: NCT05546580 .

Classification of Acute Myeloid Leukemia by Cell-Free DNA 5-Hydroxymethylcytosine.

Epigenetic abnormality is a hallmark of acute myeloid leukemia (AML), and aberrant 5-hydroxymethylcytosine (5hmC) levels are commonly observed in AML patients. As epigenetic subgroups of AML correlate with different clinical outcomes, we investigated whether plasma cell-free DNA (cfDNA) 5hmC could categorize AML patients into subtypes. We profiled the genome-wide landscape of 5hmC in plasma cfDNA from 54 AML patients. Using an unbiased clustering approach, we found that 5hmC levels in genomic regions with a histone mark H3K4me3 classified AML samples into three distinct clusters that were significantly associated with leukemia burden and survival. Cluster 3 showed the highest leukemia burden, the shortest overall survival of patients, and the lowest 5hmC levels in the TET2 promoter. 5hmC levels in the TET2 promoter could represent TET2 activity resulting from mutations in DNA demethylation genes and other factors. The novel genes and key signaling pathways associated with aberrant 5hmC patterns could add to our understanding of DNA hydroxymethylation and highlight the potential therapeutic targets in AML. Our results identify a novel 5hmC-based AML classification system and further underscore cfDNA 5hmC as a highly sensitive marker for AML.

PRMT1 inhibition promotes ferroptosis sensitivity via ACSL1 upregulation in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a hematological malignancy with an alarming mortality rate. The development of novel therapeutic targets or drugs for AML is urgently needed. Ferroptosis is a form of regulated cell death driven by iron-dependent lipid peroxidation. Recently, ferroptosis has emerged as a novel method for targeting cancer, including AML. Epigenetic dysregulation is a hallmark of AML, and a growing body of evidence suggests that ferroptosis is subject to epigenetic regulation. Here, we identified protein arginine methyltransferase 1(PRMT1) as a ferroptosis regulator in AML. The type I PRMTinhibitor GSK3368715 promoted ferroptosis sensitivity in vitro and in vivo. Moreover, PRMT1-knockout cells exhibited significantly increased sensitivity to ferroptosis, suggesting that PRMT1 is the primary target of GSK3368715 in AML. Mechanistically, both GSK3368715 and PRMT1knockout upregulated acyl-CoA synthetase long-chain family member 1(ACSL1), which acts as a ferroptosis promoter by increasing lipid peroxidation. Knockout ACSL1 reduced the ferroptosis sensitivity of AML cells following GSK3368715 treatment. Additionally, the GSK3368715 treatment reduced the abundance of H4R3me2a, the main histone methylation modification mediated by PRMT1, in both genome-wide and ACSL1 promoter regions. Overall, our results demonstrated a previously unknown role of the PRMT1/ACSL1 axis in ferroptosis and suggested the potential value and applications of the combination of PRMT1 inhibitor and ferroptosis inducers in AML treatment.

Activation of orphan receptor GPR132 induces cell differentiation in acute myeloid leukemia

Blocked cellular differentiation is a critical pathologic hallmark of acute myeloid leukemia (AML). Here, we showed that genetic activation of the orphan GPCR GPR132 significantly induced cell differentiation of AML both in vitro and in vivo, indicating that GPR132 is a potential trigger of myeloid differentiation. To explore the therapeutic potential of GPR132 signaling, we screened and validated a natural product 8-gingerol (8GL) as a GPR132 agonist. Notably, GPR132 activation by 8GL promoted differentiation and reduced colony formation in human AML cell lines with diverse genetic profiles. Mechanistic studies revealed that 8GL treatment inhibits the activation of the mammalian target of rapamycin (mTOR), a regulator of AML cell differentiation blockade, via activating GPR132-Gs-PKA pathway. We further showed that the combination of 8GL and an mTOR inhibitor synergistically elicited AML cell differentiation in vitro. Importantly, 8GL alone or in combination with an mTOR inhibitor remarkably impaired tumor growth and extended mouse survival in an AML xenograft model accompanied by enhanced cell differentiation. Notably, genetic or pharmacological activation of GPR132 triggered the differentiation of human primary AML cells. In summary, this study demonstrated that activation of orphan GPR132 represents a potential strategy for inducing myeloid differentiation in AML patients.

Inhibition of Stearoyl-CoA Desaturase Has Anti-Leukemic Properties in Acute Myeloid Leukemia

Metabolic rewiring, a hallmark of acute myeloid leukemia (AML), contributes to the development of resistance to intensive chemotherapy and has been shown to play an essential role in leukemic cell maintenance. Several metabolic alterations have been described in AML, such as dependence on amino acids to fuel oxidative phosphorylation or ability of leukemic cells to switch energy dependence to fatty acid oxidation in response to treatment. However, little is known about the functional significance of the fatty acid synthesis (FAS) pathway in AML. Here we have identified stearoyl-CoA desaturase (SCD), a key enzyme responsible for the conversion of saturated fatty acids (SFA) into monounsaturated fatty acids (MUFA), as a novel therapeutic target in AML. High SCD expression predicts worse survival across several patient datasets, even after correction for gender, age and AML European LeukemiaNet risk category. Moreover, genetic and chemical inhibition of SCD reduces growth and induces cell death in AML cells. Since targeting SCD with currently available inhibitors has been challenging because of their toxicity profile in vivo, we have accessed a novel pharmacological SCD inhibitor with a more favorable toxicity profile, SSI-4 (Modulation Therapeutics Inc., Morgantown, USA). SSI-4 reduces viability in several human and mouse AML models in vitro and in about half of the 36 primary AML samples tested ex vivo. Moreover, SSI-4 displays anti-leukemic effects in AML murine models and patient-derived xenografts (PDX). Since we could not correlate susceptibility to SCD inhibition with any genetic characteristic of AML cells, we aimed to determine the molecular basis of cell sensitivity using phosphoproteomic, proteomic and transcriptomic approaches coupled with metabolic analyses of sensitive and resistant cell lines and primary AML cells. Sensitivity to SCD inhibition directly correlates with rate of de novo FAS, as measured by fatty acid labeling with 13C-glucose. Moreover, modulation of FAS by the AMPK signaling activity dictates sensitivity to SCD inhibition and AMPK activation rescues the anti-leukemic effects of SSI-4. The anti-leukemic effects of SCD inhibition are secondary to the induction of SFA mediated lipotoxicity, as demonstrated by the increased sensitivity of previously resistant cells to SSI-4, when grown in the presence of the SFA palmitate. Conversely, growing cells in the presence of the MUFA oleate, as well as inhibition of FAS by targeting enzymes upstream of SCD, results in resistance to SSI-4, thus confirming that the increased SFA/MUFA ratio drives leukemic cell death. SFA mediated lipotoxicity leads to both increased lipid peroxidation and activation of the unfolded protein response (UPR), namely its PERK branch, which in turn leads to apoptotic cell death. Indeed, inhibition of PERK, but not of other branches of UPR, rescues SCD-mediated cytotoxicity. Despite causing disease debulking in vivo, SSI-4 does not prolong survival in murine AML models as a single agent and the toxic effects of SSI-4 are decreased when AML cells are grown in co-culture with stroma, suggesting a protective role of tumor microenvironment. However, we consistently detect increased lipid peroxidation and DNA damage both in vitro and in vivo following SCD inhibition. We therefore combined SSI-4 with doxorubicin, a known inducer of lipid peroxidation and DNA damage, which is routinely used in AML induction therapy. Indeed, both genetic depletion and pharmacological inhibition of SCD renders AML cells more sensitive to doxorubicin, and addition of SSI-4 to a standard AML chemotherapy protocol consisting of doxorubicin and cytarabine significantly extends survival in AML mouse models. In summary, we identify and validate SCD as a therapeutic target in AML. The novel SCD inhibitor SSI-4 displays anti-leukemic properties both in vitro and in vivo across different AML models and particularly when combined with standard chemotherapy while showing minimal toxicity in vivo. We show that sensitivity to SCD inhibition is driven by metabolic rather than genetic AML features, thus making this therapeutic approach potentially applicable across several AML subtypes, including high risk ones. Finally, the mechanism of toxicity following SCD inhibition suggests that this can be further enhanced by modulating the levels of SFA/MUFA through dietary interventions or by combining it with DNA damaging agents. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Frida Study: An Option for Mutated FLT3 Relapsed/Refractory Acute Myeloid Leukemia Patients with a Novel Iadademstat and Gilteritinib Combination Therapy

Background: Epigenetic dysregulation is a hallmark of acute myeloid leukemia (AML) and is caused by recurrent translocations and/or mutations in chromatin regulators and transcription factors, resulting in myeloid differentiation blockade and leukemic stem cell renewal. Accordingly, about 70% of recurring mutations in AML patients target regulators of gene expression, underscoring the potential of epigenetic therapies to change the natural history of the disease. Iadademstat (iada/ORY-1001) is a specific, oral, potent, covalent inhibitor of the epigenetic Lysine-Specific Demethylase 1 (LSD1/KDMA1) enzyme. Preclinical models demonstrate that exposure to iada decreases leukemic stem cell survival and preclinical and clinical studies shows iada's effect on inducing macrophage/monocytic differentiation of blast cells. Furthermore, a Phase 2 ongoing study of iada in combination with azacitidine (ALICE) shows high complete remission (CR)/ CR with incomplete count recovery (CRi) and durable responses in treatment naïve, unfit AML patients (pts) without exacerbating the toxicity profile of azacitidine (Salamero et al., Abstract P586 EHA 2022). Despite improvements in AML therapy, relapses remain a common challenge, contributing to the death of more than 50% of pts, particularly in those subpopulations with higher risk genetics, the largest of which (up to 30-40% of AML pts) harbor mutations in the fms-like tyrosine kinase 3 (FLT3) gene. Use of the FLT3 inhibitor (FLT3i) gilteritinib as monotherapy for relapsed/refractory (R/R) pts has resulted in improved outcomes but the duration of remission achieved is transient and often brief. The Phase 3 randomized ADMIRAL trial of gilteritinib revealed a CR rate of 20%; and an event-free-survival (EFS) of 2.8 months (Perl, et al., NEJM 2019). Preclinically, iada produces marked synergy with FLT3i, including gilteritinib, in FLT3 wild-type and mutated (FLT3 mut+) AML cells and in derived cell lines resistant to venetoclax, azacitidine and other FLT3is. The novel MOAs generating this synergy of the combination include activation of a pro-differentiative epigenetic and transcriptional program with simultaneous suppression of the activity of MYC target genes (Yashar, et al. 2022, https://doi.org/10.1101/2022.01.17.476469). The FRIDA clinical trial is designed to establish the safety, tolerability of the combination of iada plus gilteritinib and to identify the recommended phase 2 dose (RP2D) in FLT3 mut+ R/R AML specifically according to FDA's Project Optimus. Methods: Adult pts with body weight ≥50 Kg and ECOG 0-2, with FLT3 mut+ R/R AML, after 1 or 2 prior lines of therapy, will be enrolled. Up to 18 pts, in a 3+3 escalation phase, will receive iada at doses of 75 to 150 ug, orally, in 5 days ON - 2 days OFF schedule, with continuous gilteritinib, at 120 mg/day orally. In the expansion phase, up to 14 pts at the selected safe and pharmacologically active dose/s (determined based on all available data from escalation pts including PK, target engagement (TE), safety, tolerability, and emerging activity) will be enrolled. TE will be assessed with a proprietary validated chemo-probe assay for LSD1 occupancy in PBMCs. Primary endpoints of the study are safety and RP2D determination, based on the same 5 criteria outlined above. Bayesian posterior probability efficacy monitoring will be performed periodically for each dose cohort starting from the first 6 additional pts in the expansion phase (total of 12). Bayesian efficacy futility and early stopping boundary will be applied during the monitoring. Posterior probability criterion (Prob (θ>0.3) ≥ 0.60) at the end of the study will warrant additional development. The safety of the combination treatment will be also continuously evaluated during expansion following a Bayesian design stopping rule. Secondary endpoints include overall survival, EFS, CR, CR/CR with partial hematologic recovery (CRh), overall response rates, time to response, duration of response, and transfusion rate. Exploratory endpoints include measurable residual disease and gene mutational analysis. Pts who achieve a response can undergo hematopoietic stem cell transplant. The study plans to have 15 sites enrolling in the US. Additional sites will be added for a subsequent randomized controlled double-blinded FRIDA 2 study to assess the efficacy of the iada and gilteritinib combination in R/R FLT3 mut+ AML.

Epigenetic Landscape Predicts Overall Survival and Remission Duration in Adult Acute Myeloid Leukemia

INTRODUCTION: Epigenetic dysregulation is a hallmark of Acute Myeloid Leukemia (AML). Although recurrent epigenetic-related mutations have been identified, the understanding of how they affect pathophysiology and leukemogenesis remains incomplete. We used proteomics to assess the epigenetic landscape in AML and identified new prognostic phenotypes and therapeutic targets. METHODS: Using Reverse Phase Protein Array (RPPA) 405 proteins (327 total; 78 post-translational modified) were measured in 818 samples from newly diagnosed adult AML patients and normalized to CD34+ cells. These included Histone H3 total and H3K4Me 1, 2, 3, K9Me2, K27Me3, K27Ac and K36Me3 Modification Marks (HMM) expression. Categorical clinical variables were compared with Fisher's Exact with simulated p-values (10000 replicates), and continuous ones with Wilcoxon or Kruskal-Wallis tests. Univariate (UV) and Multivariate (MV) models for Overall Survival (OS) and Remission Duration (RD) were built with Cox proportional hazards model (CoxPH). Differentially Expressed Proteins were determined using the Kruskal-Wallis test (p<0.05) and pairwise Dunn's tests with p-values adjusted with the False Discovery Rate method (p<0.05). RESULTS: Using unbiased hierarchical clustering, patients were clustered into 4 groups (Fig. 1, C1 to C4) based on the expression of 2 Repressive HMM (H3K27Me3 and H3K9Me2), and 5 Activating HMM (H3K27Ac, H3K36Me3, H3K4Me3, H3K4Me2, H3K4Me1). High repression and low activation HMM were prominent in C1, while C4 had the opposite pattern. Higher expression of all marks and Total H3 defined C2, and C3 had lower expression of all marks, and Total H3. Importantly, when normalized by Total H3, HMM expression of both C2 and C3 are close to average, representing a chromatin state between C1 and C4. Compared to the other groups, patients in C4 were older (66yo vs 62yo; p=0.006), and had more secondary AML (55% vs 43%; p=0.007) and unfavorable cytogenetic risk (55% vs 37%; p<0.001). Mutations weren't exclusively associated with any cluster: ASLX1 and NPM1 mutants were more frequent in C1 and C2 (27% vs 13%; p<0.001; 23% vs 15%; p=0.015), WT1 more in C1 and C3 (9% vs 3.7%, p=0.018), and EZH2, PTPN11, RAS and RUNX1 happened more in C3 and C4 (7.7% vs 1.7%, p<0.001; 13% vs 5.6%, p=0.002; 26% vs 14%, p<0.001; 22% vs 14%, p=0.015). The effect of HMM expression on OS (Fig. 2) showed that patients with high repression and low activation (C1) did significantly better (Median OS (MS) = 19mo, Median RD (MRD)>102mo), and those with the opposite pattern of low repression and high activation (C4) did significantly worse (MS=7mo, MRD=16mo). Patients with an intermediary chromatin state were close to the average OS (C2: MS=14mo, MRD=20mo; C3: MS=12mo, MRD=24mo). In the UV CoxPH model, C1, C3 and C4 predicted OS (HR=1, 1.3, 1.4, 2.3; p=ref., 0.007, 0.001), as did age, secondary AML, complex karyotype, and CEBPA, FLT3, KIT, JAK2, NPM1, PTPN11, SRSF2 and TP53 mutations. In the MV model C1, C3 and C4 were again predictive for OS (HR=1, 1.5, 2.4, 2.7; p=ref., 0.003, 0.006), together with age, secondary AML, and PTPN11, SRSF2 and TP53 mutations. For RD, in the UV model, all clusters predicted RD (HR=1, 1.9, 1.8, 2.7; p=ref., 0.002, 0.005, 0.001), as did age, secondary AML, complex karyotype, and FLT3, NPM1, RUNX1 and TP53 mutations. In the MV model of RD, all clusters were predictors (HR=1, 1.8, 1.8, 2.7; p=ref., 0.045, 0.038, 0.003), together with age, complex karyotype and RUNX1 mutation. Differentially Expressed Proteins, from the other 397 proteins, for C4 revealed high activation of VEGF, ErbB and FcεRI signaling pathways, unveiling potential new druggable targets. CONCLUSION: HMM expression found that opposing chromatin states, with a more closed (C1) or open (C4) chromatin, significantly affected prognosis. Moreover, the epigenetic landscape was useful to predict OS and RD: compact chromatin equals high OS and RD (C1), and a loose one means poor OS and RD (C4), whereas an intermediary state (C2/C3) leads to average OS and RD. The expression pattern of the worst OS group showed high activation of specific signaling pathways which could be targeted to improve OS and RD. It would be worthwhile to correlate the findings with ATAC-seq, ChIP-seq, RNA-seq and DNA methylation analysis, in order to determine the loci responsible for the observed phenotypes in each cluster, and also WGS to identify possible new drivers of chromatin aberrations in AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Constitutional pericentric inversion of chromosome 16, inv(16)(p13.1q22), mimicking acute myeloid leukemia.

An acquired abnormality, inv(16)(p13q22), is a hallmark of acute myeloid leukemia with eosinophilia (AML M4Eo), which leads to leukemogenesis by forming the CBFB::MYH11 fusion gene [1]. We experienced a case of inv(16)(p13.1q22) discovered incidentally during bone marrow examination without evidence of AML. A 64-year-old woman was referred to our hospital because of hypergammaglobulinemia. She was being treated for rheumatoid arthritis, but had no past and family history of AML. Laboratory examination showed polyclonal gammopathy but no significant abnormalities in complete blood count with differential. Bone marrow picture showed a normocellular marrow and mild increase in plasma cells to 3.8% (reference range, 0.2%–1.7%), but no evidence of hematological malignancy. Surprisingly, chromosome analysis of the bone marrow cells revealed that all 20 cells had inv(16)(p13.1q22) mimicking AML M4Eo (Figure 1A). We therefore performed fluorescence in situ hybridization (FISH) analysis of the bone marrow cells by using the Vysis CBFB Dual Color Break Apart Rearrangement Probe (Abbott Molecular Diagnostics, Des Plaines, IL, USA). The splitting of the red and green signals reflecting truncation of the CBFB locus on chromosome 16q22 was not observed (Figure 1B). In addition, a multiplex reverse transcriptase-polymerase chain reaction assay covering 28 leukemia-specific fusion transcripts including CBFB::MYH11 was performed by using the HemaVision-28Q kit (VERITAS, Tokyo, Japan), but none of them was detected. Therefore, it was suggested that the chromosome inversion of this patient did not disrupt the CBFB gene. High-resolution chromosome analysis of the phytohemagglutinin (PHA)-stimulated peripheral blood lymphocytes also revealed inv(16)(p13.1q22) in all 20 cells, indicating a constitutional abnormality of chromosome 16 (Figure 1C). Thus, this patient was not misdiagnosed as AML M4Eo and has been followed up without treatment. To the best of our knowledge, only a few cases with inv(16)(p13q22) unrelated to AML have been reported [2-5]. All of these were concluded to be constitutional abnormalities, and some cases have been confirmed as familial onset. In these reports, there was no misdiagnosis of AML M4Eo in individuals without hematological malignancy, and in the case of chronic myeloid leukemia with t(9;22)(q34;q11) and inv(16)(p13q22), FISH analysis showed no abnormality of the MYH11 gene and inv(16)(p13q22) was also found in her healthy father, which avoided misdiagnosis as an additional cytogenetic abnormality in chronic phase [4]. Therefore, although extremely rare, we should be aware of the presence of constitutional chromosome abnormalities mimicking hematological malignancies, and it is important to perform further tests such as FISH and genomic analysis for differential diagnosis. We thank Yoshiko Sato, MT (LSI Medience Corporation, Tokyo, Japan), for her technical support. The authors declare that there is no conflict of interest. All procedures in this study were performed in accordance with the principles of the Declaration of Helsinki and the institutional guidelines. Informed consent was obtained from the patient and her family. The data that support the findings of this study are available from the corresponding author upon reasonable request.

Disruption of dNTP homeostasis by ribonucleotide reductase hyperactivation overcomes AML differentiation blockade

AbstractDifferentiation blockade is a hallmark of acute myeloid leukemia (AML). A strategy to overcome such a blockade is a promising approach against the disease. The lack of understanding of the underlying mechanisms hampers development of such strategies. Dysregulated ribonucleotide reductase (RNR) is considered a druggable target in proliferative cancers susceptible to deoxynucleoside triphosphate (dNTP) depletion. Herein, we report an unanticipated discovery that hyperactivating RNR enables differentiation and decreases leukemia cell growth. We integrate pharmacogenomics and metabolomics analyses to identify that pharmacologically (eg, nelarabine) or genetically upregulating RNR subunit M2 (RRM2) creates a dNTP pool imbalance and overcomes differentiation arrest. Moreover, R-loop–mediated DNA replication stress signaling is responsible for RRM2 activation by nelarabine treatment. Further aggravating dNTP imbalance by depleting the dNTP hydrolase SAM domain and HD domain-containing protein 1 (SAMHD1) enhances ablation of leukemia stem cells by RRM2 hyperactivation. Mechanistically, excessive activation of extracellular signal-regulated kinase (ERK) signaling downstream of the imbalance contributes to cellular outcomes of RNR hyperactivation. A CRISPR screen identifies a synthetic lethal interaction between loss of DUSP6, an ERK-negative regulator, and nelarabine treatment. These data demonstrate that dNTP homeostasis governs leukemia maintenance, and a combination of DUSP inhibition and nelarabine represents a therapeutic strategy.

An oncogenic enhancer encodes selective selenium dependency in AML.

Deregulation of transcription is a hallmark of acute myeloid leukemia (AML) that drives oncogenic expression programs and presents opportunities for therapeutic targeting. By integrating comprehensive pan-cancer enhancer landscapes with genetic dependency mapping, we find that AML-enriched enhancers encode for more selective tumor dependencies. We hypothesized that this approach could identify actionable dependencies downstream of oncogenic driver events and discovered a MYB-regulated AML-enriched enhancer regulating SEPHS2, a key component of the selenoprotein production pathway. Using a combination of patient samples and mouse models, we show that this enhancer upregulates SEPHS2, promoting selenoprotein production and antioxidant function required for AML survival. SEPHS2 and other selenoprotein pathway genes are required for AML growth invitro. SEPHS2 knockout and selenium dietary restriction significantly delay leukemogenesis invivo with little effect on normal hematopoiesis. These data validate the utility of enhancer mapping in target identification and suggest that selenoprotein production is an actionable target in AML.

The retinoic acid receptor co-factor NRIP1 is uniquely upregulated and represents a therapeutic target in acute myeloid leukemia with chromosome 3q rearrangements

Aberrant expression of Ecotropic Viral Integration Site 1 (EVI1) is a hallmark of acute myeloid leukemia (AML) with inv(3) or t(3;3), which is a disease subtype with especially poor outcome. In studying transcriptomes from AML patients with chromosome 3q rearrangements, we identified a significant upregulation of the Nuclear Receptor Interacting Protein 1 (NRIP1) as well as its adjacent non-coding RNA LOC101927745. Utilizing transcriptomic and epigenomic data from over 900 primary samples from patients as well as genetic and transcriptional engineering approaches, we have identified several mechanisms that can lead to upregulation of NRIP1 in AML. We hypothesize that the LOC101927745 transcription start site harbors a context-dependent enhancer that is bound by EVI1, causing upregulation of NRIP1 in AML with chromosome 3 abnormalities. Furthermore, we showed that NRIP1 knockdown negatively affects the proliferation and survival of 3qrearranged AML cells and increases their sensitivity to all-trans retinoic acid, suggesting that NRIP1 is relevant for the pathogenesis of inv(3)/t(3;3) AML and could serve as a novel therapeutic target in myeloid malignancies with 3q abnormalities.