Maintenance Of Acute Myeloid Leukemia Research Articles

Clinical activity, pharmacokinetics, and pharmacodynamics of oral hypomethylating agents for myelodysplastic syndromes/neoplasms and acute myeloid leukemia: A multidisciplinary review.

To review the pharmacokinetic (PK)-pharmacodynamic (PD) profiles, disease setting, dosing, and safety of oral and parenteral hypomethylating agents (HMAs) for the treatment of myelodysplastic syndromes/neoplasms (MDS) and acute myeloid leukemia (AML), and to provide a multidisciplinary perspective on treatment selection and educational needs relating to HMA use. Clinical and real-world data for parenteral decitabine and azacitidine and two oral HMAs: decitabine-cedazuridine (DEC-C) for MDS and azacitidine (CC-486) for AML maintenance therapy. Differences in the PK-PD profiles of oral and parenteral HMA formulations have implications for their potential toxicities and planned use. Oral DEC-C (decitabine 35 mg and cedazuridine 100 mg) has demonstrated equivalent systemic area under the concentration-time curve (AUC) exposure to a 5-day regimen of intravenous (IV) decitabine 20 mg/m2 and showed no significant difference in PD. The AUC equivalence of oral DEC-C and IV decitabine means that these regimens can be treated interchangeably (but must not be substituted within a cycle). Oral azacitidine has a distinct PK-PD profile versus IV or subcutaneous azacitidine, and the formulations are not bioequivalent or interchangeable owing to differences in plasma time-course kinetics and exposures. Clinical trials are ongoing to evaluate oral HMA combinations and novel oral HMAs, such as NTX-301 and ASTX030. Treatment with oral HMAs has the potential to improve quality of life, treatment adherence, and disease outcomes versus parenteral HMAs. Better education of multidisciplinary teams on the factors affecting HMA treatment selection may help to improve treatment outcomes in patients with MDS or AML.

TMIGD2 is an orchestrator and therapeutic target on human acute myeloid leukemia stem cells

Acute myeloid leukemia (AML) is initiated and sustained by a hierarchy of leukemia stem cells (LSCs), and elimination of this cell population is required for curative therapies. Here we show that transmembrane and immunoglobulin domain containing 2 (TMIGD2), a recently discovered co-stimulatory immune receptor, is aberrantly expressed by human AML cells, and can be used to identify and enrich functional LSCs. We demonstrate that TMIGD2 is required for the development and maintenance of AML and self-renewal of LSCs but is not essential for normal hematopoiesis. Mechanistically, TMIGD2 promotes proliferation, blocks myeloid differentiation and increases cell-cycle of AML cells via an ERK1/2-p90RSK-CREB signaling axis. Targeting TMIGD2 signaling with anti-TMIGD2 monoclonal antibodies attenuates LSC self-renewal and reduces leukemia burden in AML patient-derived xenograft models but has negligible effect on normal hematopoietic stem/progenitor cells. Thus, our studies reveal the function of TMIGD2 in LSCs and provide a promising therapeutic strategy for AML.

Identification of a Selective FLT3 Inhibitor with Low Activity against VEGFR, FGFR, PDGFR, c-KIT, and RET Anti-Targets.

FLT3 is mainly expressed in immune and various cancer cells and is a drug target for acute myeloid leukemia (AML). Recently, FLT3 has also been identified as a potential target for treating chronic pain. Most FLT3 inhibitors (FLT3i) identified to date, including approved drugs such as gilteritinib, midostaurin, ponatinib, quizartinib, and FLT3i in clinical trials, such as quizartinib and crenolanib, also inhibit closely-related kinases that are important for immune (c-KIT), cardiovascular (KDR/VEGFR2, FGFR, PDGFR) or kidney (RET) functions. While the aforementioned FLT3i may increase survival rates in AML, they are neither ideal for AML maintenance therapy nor for non-oncology applications, such as for the treatment of chronic pain, due to their promiscuous inhibition of many kinase anti-targets. Here, we report the identification of new FLT3i compounds that have low activities against kinases that have traditionally been difficult to differentiate from FLT3 inhibition, such as KDR/VEGFR, FGFR, PGFR, c-KIT, and RET. These selective compounds could be valuable chemical probes for studying FLT3 biology in the context of chronic pain and/or may represent good starting points to develop well-tolerated FLT3 therapeutics for non-oncology indications or for maintenance therapy for AML.

PRMT1 Promotes Chronic Lymphocytic Leukemia Progression Via Modulating Methylation of MAST1

Introduction As a member of the protein arginine N-methyltransferase family, protein arginine methyltransferase 1 (PRMT1) plays a crucial regulatory role in the etiology of malignant neoplasms. PRMT1-mediated protein methylation promoted the maintenance of acute myeloid leukemia, which revealed the promising potency of PRMT1 inhibitors. Recent study suggested that dysregulation of PRMT5 promotes Richter's transformation in chronic lymphocytic leukemia (CLL). However, the effects of PRMT1 in the tumorigenesis and progression of CLL still remained ill-defined. Hence, the aim of this study was to investigate the clinical significance and mechanisms of PRMT1 underlying the development of CLL. Methods Peripheral blood samples were collected from 79 newly diagnosed CLL patients (47 males and 32 females; age range 39-85 years, median 63 years) in Shandong Provincial Hospital CLL (SPHCLL) cohort with informed consent. CRISPR-Cas9 technology was used to stably knockout PRMT1 in CLL cells. A label free quantitative proteomics analysis was implemented to reveal the protein methylation mediated by PRMT1. Assessment of cell viability, apoptosis and cell cycle were analyzed by cell counting kit-8, annexin V-PE/7AAD and PI/ RNase staining, respectively. This study was approved by the Medical Ethics Committee of Shandong Provincial Hospital. Results This study examined the expression of PRMT1 in SPHCLL and GEO databases, and discovered the upregulation of PRMT1 mRNA in CLL cells. Aberrantly elevated expression of PRMT1 was observed in a cohort of newly diagnosed CLL patients than healthy donors in mRNA level (donors vs. CLL patients, 0.07±0.06 vs. 0.20 ±0.21, p=0.013). Furthermore, increased expression of PRMT1 was correlated with inferior prognosis in two long-term follow-up cohorts of CLL patients (HR=2.80, p=0.002, and HR=1.551, p=0.009). To elucidate the functional significance of PRMT1 in CLL, we established stable PRMT1 knockdown cells using lentiviral shRNAs and PRMT1 knockout cells using CRISPR/Cas9 technology. The suppression of PRMT1 remarkably inhibited cell proliferation, induced cell apoptosis and blocked cell cycle at G1/S phase in CLL cells. Furthermore, it was observed that C7280948, a selective inhibitor of PRMT1, resulted in the defective proliferation of CLL primary cells in a dose-dependent manner. We subsequently performed pre-clinical investigations of C7280948 in the orthotopic CLL xenograft murine model. Notably, the administration of C7280948 significantly diminished the leukemia burden and induced the lessened degree of splenomegaly in comparison to the control group (Fig. 1A). Moreover, a reduction in the population of CLL cells was detected in bone marrow and spleen of mice treated with C7280948. To decipher the role of PRMT1 in the pathogenesis of CLL, the label free quantitative proteomics analysis was conducted on PRMT1-deficent and control MEC1 cells. Knockdown of PRMT1 diminished asymmetric dimethylarginine of some proteins in CLL cells. In accordance with the results of proteomics analysis, microtubule associated serine/threonine kinase 1 (MAST1) was selected as a candidate target for further investigation. The methylation site of MAST1 protein was identified as R802 (Fig. 1B). Increased expression of MAST1 was correlated with inferior prognosis of CLL patients ( p<0.001), indicating the essential role of MAST1 in the progression of CLL. These results supported the hypothesis that PRMT1 interacted with MAST1 and enhanced the activity of MAST1 via methylating this oncoprotein in CLL cells. Conclusion The present study provides robust evidence for the oncogenic function of PRMT1 in the pathogenesis of CLL, highlighting the potential therapeutic efficacy of the selective PRMT1 inhibitor C7280948. Collectively, the targeted inhibition of PRMT1 exhibits promising prospects for the management of CLL patients.

Treatment of Elderly Patients with Higher Risk Myelodysplastic Syndromes with Oral Azacitidine

Background Oral formulations ofhypomethylating agents decitabine (ASTX727) and azacitidine (cc-486, AZA) were approved respectively for treatment of higher risk myelodysplastic syndromes (HR-MDS) and for maintenance of acute myeloid leukemia after remission. Management of MDS patients with oral agents is relevant to improve quality of life. Oral-AZA has shown activity in lower risk MDS and its use is under evaluation in that setting. There are no data regarding maintenance of response achieved by subcutaneous (sc) AZA in HR-MDS cases by switching to the oral formulation. Aim The primary objective of the study was to explore the feasibility of replacing sc-AZA by its oral formulation in patients with HR-MDS in response. Maintenance or improvement of response and, as secondary objectives, patient reported outcome (PRO) and DNA methylation pattern were evaluated. Methods A monocentric, pilot phase 2 study was planned to enroll 11 subjects with confirmed diagnosis of MDS, IPSS-R higher risks, aged ≥ 65 years, in CR/CRi, PR or SD with HI after > 6 cycles of sc-AZA therapy (ClinicalTrial.gov NCT04806906). Patients were to receive 300 mg oral-AZA for the first 14-days of each 28-day treatment cycle until absence of benefit or disease progression. Dose of oral-AZA could be de-escalated based on toxicity. At screening and every 4 cycles, IWG response was evaluated. NGS evaluation of somatic mutations and DNA methylation analysis were performed at the same time points. DNA methylation pattern of separated BM CD34 positive cells was determined by Oxford Nanopore. At day1 of every cycle the EQ-5D questionnaire for QoL was administered. Feasibility, safety, tolerability as well as efficacy of oral-AZA were evaluated by monitoring AEs and response/loss of response, time to treatment discontinuation, and PRO. Results. As per July 31 st 2023, 11 HR-MDS patients with a median follow up of 167 days (range 47-805) were enrolled in this study. Male/female ratio was 4.5/1 with a median age of 81 yrs (67-88). At diagnosis patients belonged to IPSS-R risk categories intermediate 1/11, high 8/11, very high 2/11; IPSS-M risk categories at start of oral AZA: 3/11 very high, 6/11 high and 2/11 very low. Median number of sc-AZA cycles was 9 (range 7-52), while for oral-AZA median number of cycles was 4 (range 1-10). Oral AZA dose of 300 mg/day was maintained for all patients. DNA methylation was determined with success at baseline and every 4 cycles for all treated patients, and methylation status analyzed. None of the patients had serious adverse events related to study drug. Myelosuppressive effects were transient and Grade1/2. Patients maintained CR (4/11), PR (6/11) and SD with HI (1/11) achieved with sc-AZA, until progression. Therapy was interrupted for progression in 4 patients who evolved to AML after a median of 9 cycles of treatment with oral-AZA. Early discontinuation was experienced in 2/11 patients: in one case following a GI event grade 3 and patient decision to avoid de-escalation of the dose for subsequent cycles, in the second case because of patient reduced compliance. Both patients re-switched from oral to sc-AZA treatment and are still in response (PR). PRO analyses indicated that oral-AZA therapy improved most quality-of-life domains compared to sc-AZA. Treatment is ongoing for 4/11 patients in CR (3/4) and PR (1/4). Responses were observed across all IPSS-M risk categories. Conclusions Treatment of HR-MDS elderly patients with oral-AZAis feasible and effective. We observed duration of hematological response with a length consistent with what shown in this patient subgroup, and even after a very prolonged treatment with sc-AZA. Oral AZA was generally well tolerated and AEs did not differ from those observed for sc-AZA.

Differentiation of Acute Myeloid Leukemia Cells upon Pharmacological Inhibition of LSD1 Requires Its N-Terminal Intrinsically Disordered Region

Lysine-specific histone demethylase 1 (LSD1/KDM1A) governs hematopoiesis by regulating hematopoietic stem cell renewal and proper differentiation. Additionally, LSD1 is required for the maintenance of acute myeloid leukemia (AML) cells and has emerged as a key therapeutic target. Prior work from our group and others have revealed that the LSD1 demethylase activity is not required for AML proliferation. Instead, the interaction between LSD1 and GFI1/GFI1B is necessary to sustain AML cell survival. Thus, beyond its canonical demethylase activity, LSD1 has nonenzymatic, transcription factor (TF)-specific scaffolding functions critical for its function. Most LSD1 inhibitors function by blocking protein-protein interactions between LSD1 and TFs, even though they were originally designed as enzyme inhibitors. While studies indicate that LSD1 has important scaffolding functions, how LSD1 can coordinate with TFs to control gene expression programs is not well understood. Here, we used CRISPR-suppressor scanning to identify regions of LSD1 outside the active site that when mutated can lead to resistance to the covalent LSD1 inhibitor, GSK-LSD1. The understudied N-terminal intrinsically disordered region (IDR) of LSD1 was among the most enriched regions. To explore this further, we generated clonal AML cell lines harboring in-frame deletions within the IDR. These IDR-mutant cell lines were drug resistant and failed to differentiate upon GSK-LSD1 treatment. Moreover, knockdown of endogenous LSD1 followed by overexpression of LSD1 IDR-mutants rescued growth in the presence of GSK-LSD1, confirming that a fully functional IDR is necessary for differentiation. We performed LSD1 co-immunoprecipitation followed by mass spectrometry in wild-type and IDR-mutant cells to measure changes in LSD1 interactors upon GSK-LSD1 treatment. GSK-LSD1 treatment disrupted the LSD1-GFI1B interaction in both wild-type and mutant cells at comparable levels. Since GSK-LSD1 still induces dissociation of the mutant complex but has minimal effect on proliferation, our results suggest that the IDR-mutant cells no longer require GFI1B. In support, knockout of GFI1B inhibited growth of wild-type but not IDR-mutant cells. Strikingly, GSK-LSD1 treatment promoted LSD1's interaction with several master regulator TFs in hematopoiesis, including C/EBPα, PU.1, and RUNX1. Notably, C/EBPα was the most up-regulated interactor in both cell lines, with modest enrichment in IDR-mutant over wild-type. Using live-cell imaging assays, we found that deletions within the LSD1 IDR enhance association with these TFs. These results suggest that GSK-LSD1 reprograms LSD1-TF interactions, disrupting LSD1-GFI1B to induce LSD1-C/EBPα association, and that the IDR mutations alters this LSD1 redistribution to other TF partners. LSD1 is known to silence gene enhancers during differentiation in a process termed “enhancer decommissioning”. The mechanisms by which LSD1 occupies and controls enhancers remain elusive. To test if the LSD1 IDR regulates enhancer elements, we profiled LSD1 and H3K27ac using ChIP-seq in wild-type and IDR-mutant cells upon GSK-LSD1 treatment. Upon GSK-LSD1 treatment, disruption of LSD1-GFI1B binding was accompanied by increased LSD1 levels at other TF and H3K27ac sites, supporting the notion that loss of LSD1-GFI1B interaction induces LSD1 redistribution to other TF partner sites. A concomitant increase in H3K27ac levels was observed across LSD1 sites co-bound by C/EBPα, PU.1, or RUNX1 in wild-type cells. By contrast, in IDR-mutant cells, increases in H3K27ac levels were blocked at these sites upon GSK-LSD1 treatment, suggesting that full enhancer activation is impaired by mutations in the IDR, potentially due to altered association with master regulator TFs. This points to a function of LSD1 IDR in controlling enhancer activation for differentiation. Collectively, our study reveals a role for IDR in regulating LSD1-TF interactions, controlling enhancer activation that is necessary for AML cell differentiation. These data provide deep functional and mechanistic insights into the role of LSD1 IDR and offer important considerations for pharmacological targeting of protein-protein interactions.

Protective Function of Human Skin Mesenchymal Niches for Acute Myeloid Leukemia-Initiating Stem Cells

Leukemia cutis (LC) or leukemic cell infiltration in skin is one of the common extramedullary manifestations in acute myeloid leukemia (AML) and signifies a poorer prognosis. However, the pathogenesis and maintenance of LC have been understudied, limiting our understanding of its impact on AML progression and relapse. We recently have shown that the AML cells infiltrated in the mouse skin during steady state and after chemotherapy were capable to regenerate AML after serial transplantation (Sandhow et al., J. Exp. Med, 2023). Further characterization of the skin mesenchymal stem cells (MSCs) suggests that skin MSCs could maintain AML-initiating stem cells (LSCs) and protect them from chemotherapy, even to a greater degree than their bone marrow (BM) counterparts. These data suggest the protective role of skin mesenchymal niche in AML LSCs during chemotherapy. However, it remains unexplored whether human skin MSCs exert the same functional impact on human AML cells. We here have explored the role of human skin MSCs in AML maintenance and survival during chemotherapy by RNA sequencing and in vitro co-culture of patient-derived AML cells with primary human skin MSCs in comparison with BM MSCs. We first by prospective characterization identified human skin MSCs with a phenotype of CD45-CD235a-CD31-CD44+CD146- from healthy donors. RNA sequencing and Q-PCR revealed expression of key hematopoietic stem cell (HSC) regulatory genes including THPO, SCF, CXCL12 and LAMA4 in human skin MSCs. Interestingly, while HSC maintenance genes such as THPO and KITLG were significantly upregulated in skin MSCs (p<0.0001 and p<0.05, respectively), HSC negative regulator SPP1 was downregulated (p=0.05), pointing to a possible better hematopoiesis-supportive function of skin MSCs. In line with this, our functional studies indicated that skin MSCs displayed a superior protective effect on human AML cell line THP-1 than their BM cell counterparts during cytarabine (Ara-C) treatment in the in vitro co-culture. There were more residual chemo-resistant AML cells, reflected in the higher number of CD36+ AML cells after treatment with Ara-C in cocultures with skin MSCs, compared to that with BM MSCs (p=0.0065). This result was confirmed by functional assays showing the increased number of cobblestone area-forming cells (p=0.0298) and colony-forming units (p=0.038) within the residual AML cells co-cultured with skin MSCs. To investigate the mechanisms involved in the protective function of human skin MSCs, we have performed RNA-sequencing of the human skin and BM MSCs before and after co-culture with AML cells. This has revealed potential unique molecular pathways between AML and skin MSCs. Furthermore, there were dramatically less altered genes in the skin MSCs compared to BM MSCs after 3 day-coculture with THP1 AML cells (221 vs 1761 altered genes with adjusted p values <0.05). These data indicate that skin MSCs are likely more resistant to AML-remodeling, which could in turn maintain AML cells in quiescence status, thereby protecting AML cells from Ara-C treatment. This speculation is currently under validation. In addition, gene set enrichment analysis showed upregulated genes associated with fatty acid metabolisms and ribosome in skin MSCs compared to BM MSCs, providing molecular evidence for a potential better metabolic support of skin MSCs for AML cells. Taken together, our preliminary data indicate an important role of human skin MSCs in supporting and protecting AML cells during Ara-C treatment. We are further investigating the underlying mechanisms.

Practice Patterns of Transplant Centers Regarding Maintenance Treatment Post Allogeneic Hematopoietic Cell Transplantation in Acute Myeloid Leukemia: A Survey on Behalf of the EBMT Acute Leukemia Working Party

Introduction: Disease recurrence in acute myeloid leukemia (AML) remains the major reason of allogeneic hematopoietic cell transplantation (allo-HCT) failure. Strategies to reduce the risk of relapse such as prophylactic pharmacologic interventions post allo-HCT are more frequently applied by some transplant physicians, especially after the growing evidence of its efficacy and tolerability. Others are still being skeptical regarding its implementation due to difficulty proving efficacy by large randomized trials, or concerns of long-term toxicities. The aim of this study was to conduct a survey among the European Society for Blood and Marrow Transplantation (EBMT)-affiliated transplant centers to identify clinical practice patterns regarding maintenance treatment post-allo-HCT in AML. Methods: After the approval of the board of the Acute Leukemia Working Party (ALWP), EBMT-affiliated centers received by email a questionnaire-based survey between January and June 2023. The questionnaire consisted of 13 questions assessing clinical practices regarding maintenance treatment post allo-HCT in AML, specifically the indications of maintenance, the type, the drug used, and the duration of the treatment. Results: A total of 115 (out of 424) EBMT-affiliated centers from 31 countries responded to the survey with a response rate of 27.12%. Overall, 102 (88.8%) centers implement maintenance strategy whether pharmacological intervention or cellular therapy post allo-HCT in AML, most of the centers (N=67, 58.3%) use it based on a personalized decision, 17 (14.8%) use it routinely, and 18 (15.7%) use it exceptionally. Regarding the indication, the majority of the centers recommend maintenance treatment for AML patients in second remission or beyond (N=64, 62.7%) or in AML patients in first remission with high risk of relapse based on cytogenetic or molecular data (N=61, 59.8%), whereas 34 (33.3%) centers recommend it only for FLT3-mutant disease, and 11 (10.8%) centers in all patients regardless of the risk of disease. The majority of centers (N=77, 75.5%) use a combination of pharmacologic and cellular therapies, and 20 (19.6%) centers use only pharmacologic therapies. Among centers using pharmacologic therapies, 62 (63.9%) use hypomethylating agents (HMA) (azacitidine N=51 (82.3%), decitabine N=6 (9.6%), HMA with venetoclax N=23 (37%), HMA with donor lymphocyte infusion (DLI) N=38 (61%), or a combination of modalities in 42 (67.7%)). In general, most centers (N=35; 87.5%) do it pre-emptively either minimal residual disease (MRD) driven or chimerism driven, whereas 22 (55%) centers do it prophylactically. The majority of centers continue HMA for one-year duration (N=37, 59.7%), 15 (24.2%) for two years and only 10 (16.1%) continue until disease recurrence or toxicity. Out of 97 responding centers regarding the use of FLT3 inhibitors as maintenance, 93 (95.9%) are implementing this strategy. The most common choice was sorafenib in 82/93 (88.2%) centers, midostaurin in 28 (30%), and gilteritinib in 30 (32.2%). Among these, 56 (83.5%) centers use FLT3 inhibitors as prophylaxis whereas 28 (41.7%) use a pre-emptive approach. The preferred maintenance duration was for two years in 51 (54.8%) centers, followed by one-year duration in 30 (32.3%) centers, continuous until disease relapse or toxicity in 9 (9.7%) centers, and for five years in only 3 (3.2%) centers. Novel agents are used in 71 (70.2%) centers as maintenance either alone or in combination with HMA, of them 52 (73.2%) use venetoclax, and 13 (13.2%) use enasidenib. Finally, regarding practice patterns, 32 (29.6%) centers claim that maintenance treatment is part of their routine practice, 46 (42.6%) only for FLT3 mutant AML. Others are not routinely implementing maintenance because they are still skeptical and need more evidence by randomized clinical trials (N=8), whereas 5 centers are concerned regarding tolerability, and 16 (14.8%) cannot implement because of regulatory restrictions or lack of access. Conclusion: In this survey, the majority of responding EBMT-affiliated transplant centers is implementing post-transplant AML maintenance treatment, predominantly HMA and FLT3 inhibitors. Further studies are needed to clarify the appropriate strategy and duration of maintenance therapy. The study will help the EBMT to incorporate post-transplant strategies as a routine registry capture parameter.

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.

COX4I1 Controls Mitochondrial Electron Transport Chain Complex IV Assembly and Leukemia Progression in Acute Myeloid Leukemia

Aberrant cellular signaling plays a pivotal role in the pathogenesis of acute myeloid leukemia (AML). Identifying key signaling components and their dependencies is crucial for advancing AML therapy. Here, we conducted a focused CRISPR library screen targeting 427 cell signaling-related genes with 3,416 sgRNAs (8 sgRNAs per gene) in the Cas9-expressing Molm13 AML cells. In addition to the known leukemia essential genes (MYC, RPS6, CCND3, GAB2, and AURKB), we discovered COX4I1 (Cytochrome c oxidase subunit 4 isoform 1) as a novel essential gene for AML maintenance (Fig. 1). CRISPR-knockout of COX4I1 in AML cells led to inhibited cell proliferation, reduced colony formation, and attenuated leukemia progression in vivo. Transcriptomic profiling revealed elevated mitochondrial stress upon COX4I1 knockout, accompanied by dysregulated mitochondrial cristae structure observed through transmission electron microscopy. Functional assays demonstrated impaired mitochondrial respiration (oxygen consumption rate, i.e., OCR, measured by Seahorse XF) and depleted cellular ATP (measured by metabolomic mass spectrometry) upon COX4I1 knockout in AML cells, highlighting the pivotal role of COX4I1 in mitochondrial homeostasis and energy metabolism. COX4I1 is a nuclear-encoded mitochondrial protein located on the mitochondrial inner membrane. Using a high-density CRISPR gene tiling scan (Fig. 2; targeting density ~2.1 aa per sgRNA), we identified two critical COX4I1 peptide regions (P1: K67 - L73 and P2: E136 - K169) required for leukemia cell proliferation and mitochondrial respiration. Mechanistically, we found that expression of COX4I1 is essential for the assembly of mitochondrial complex IV, a crucial component of the electron transport chain involved in establishing the proton electrochemical potential across the mitochondrial inner membrane. Structural analysis revealed that facing the mitochondrial matrix, COX4I1's P1 region establishes the interface between COX4I1 and COX5A, which is necessary for the formation of the COX1 module. Conversely, COX4I1's P2 region (C-terminal 34 residues) is dispensable to the COX1 module formation; nonetheless, the P2 region co-localizes with the intermembrane space domains of the COX2/COX3 modules and is required for the assembling of an intact complex IV. Finally, we found that knockout of COX4I1 induced mitochondrial stress further triggers cellular ferroptosis in AML cells, which is accompanied by an imbalanced cellular redox index (reduced GSH/GSSG ratio) and elevated lipid peroxidation (increased malondialdehyde). These notions led us to discover the critical role of COX4I1 as an opportunity for targeted AML therapy, especially when combined with mitochondria-targeting therapeutics such as Venetoclax (i.e., ABT-199). In conclusion, our study highlights COX4I1 as a novel vulnerability in AML, controlling mitochondrial complex IV assembly and ATP biosynthesis. This finding holds promise for improving AML therapy and may be extended to identify therapeutically relevant signaling components in other hematopoietic malignancies.

MiR-30e-5p regulates leukemia stem cell self-renewal through the Cyb561/ROS signaling pathway.

Leukemia stem cells (LSC) represent a crucial and rare subset of cells present in acute myeloid leukemia (AML); they play a pivotal role in the initiation, maintenance, and relapse of this disease. Targeting LSC holds great promise for preventing AML relapse and improving long-term outcomes. However the precise molecular mechanisms governing LSC self-renewal are still poorly understood. Here, we present compelling evidence that the expression of miR-30e-5p, a potential tumor-suppressive microRNA, is significantly lower in AML samples than in healthy bone marrow samples. Forced expression of miR- 30e effectively inhibits leukemogenesis, impairs LSC self-renewal, and delays leukemia progression. Mechanistically, Cyb561 acts as a direct target of miR-30e-5p in LSC, and its deficiency restricts the self-renewal of LSC by activating reactive oxygen series signaling and markedly prolongs recipients' survival. Moreover, genetic or pharmacological overexpression of miR-30e-5p or knockdown of Cyb561 suppresses the growth of human AML cells. In conclusion, our findings establish the crucial role of the miR-30e-5p/Cyb561/ROS axis in finely regulating LSC self-renewal, highlighting Cyb561 as a potential therapeutic target for LSC-directed therapies.

Deubiquitinase USP9X stabilizes RNA m6A demethylase ALKBH5 and promotes acute myeloid leukemia cell survival

Post-translational modifications including protein ubiquitination regulate a plethora of cellular processes in distinct manners. RNA N6-methyladenosine is the most abundant post-transcriptional modification on mammalian mRNAs and plays important roles in various physiological and pathological conditions including hematologic malignancies. We previously determined that the RNA N6-methyladenosine eraser ALKBH5 is necessary for the maintenance of acute myeloid leukemia (AML) stem cell function, but the post-translational modifications involved in ALKBH5 regulation remain elusive. Here, we show that deubiquitinase ubiquitin-specific peptidase 9X (USP9X) stabilizes ALKBH5 and promotes AML cell survival. Through the use of mass spectrometry as an unbiased approach, we identify USP9X and confirm that it directly binds to ALKBH5. USP9X stabilizes ALKBH5 by removing the K48-linked polyubiquitin chain at K57. Using human myeloid leukemia cells and a murine AML model, we find that genetic knockdown or pharmaceutical inhibition of USP9X inhibits leukemia cell proliferation, induces apoptosis, and delays AML development. Ectopic expression of ALKBH5 partially mediates the function of USP9X in AML. Overall, this study uncovers deubiquitinase USP9X as a key for stabilizing ALKBH5 expression and reveals the important role of USP9X in AML, which provides a promising therapeutic strategy for AML treatment in the clinic.

Mutant NPM1: Nuclear export and the mechanism of leukemogenesis.

Mutant NPM1: Nuclear export and the mechanism of leukemogenesis.

SOHO State of the Art Updates and Next Questions |The Role of Maintenance Therapy in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is an aggressive disease predominantly affecting the elderly population. Although, up to 65% of patients with AML achieve a complete remission with standard induction chemotherapy, the majority of patients will relapse and succumb to the disease. Although maintenance therapy is a component of standard management for various hematological malignancies, such as acute lymphoblastic leukemia (ALL), acute promyelocytic leukemia (APL) or multiple myeloma, past studies investigating the role of maintenance therapy in AML were unable to demonstrate an advantage in overall survival, and therefore, it has not been an established practice in the treatment of AML. For patients, who are not candidates for stem cell transplant, effective AML maintenance therapies are needed in order to reduce the risk of relapse. Over the past decades, many investigators have examined the role of various maintenance strategies in AML; with the intention to prolong remission and overall survival. This review will provide an overview of prior and ongoing approaches and strategies to maintenance therapy for AML.

Novel First-in-Class Drug ONC201 As a Post-Transplant Maintenance for AML and MDS: A Phase I Trial in Progress

Background: Relapses of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) after allogeneic hematopoietic cell transplantation represent an area of significant unmet clinical need and a research priority identified by the National Cancer Institute. For patients with high-risk AML and MDS, post-transplant relapses remain the principal cause of mortality. The role of chemotherapy agents outside of FLT3 inhibitors or TKIs, and post-transplant cellular therapy to prevent relapse of leukemia are not well established. However, chemotherapy agents are frequently utilized off-label in clinical practice, thus signifying an urgent need to develop effective therapeutic strategies. ONC201 is a novel first-in-class small molecule imipridone, that antagonizes G protein-coupled receptor, DRD2 resulting in AKT/ERK inactivation. ONC201 is also an allosteric agonist of mitochondrial caseinolytic protease P that activates an integrated stress responses. In preclinical studies, leukemia cells demonstrate sensitivity to ONC201 regardless of their genetic and mutation profiles. Leukemia cells with genetic and mutation changes such as TP53 mutation and complex karyotype that confer resistance to conventional therapy are sensitive to ONC201. ONC201 is also toxic to leukemia stem cells while sparing normal bone marrow cells [Sci Signal. 2016 Feb 16;9(415):ra17]. Several clinical trials in solid and hematologic malignancies have demonstrated that oral ONC201 has a low toxicity profile and is well tolerated. The profile of ONC201 is well suited for further development to prevent post-transplant relapses of AML and MDS. Here, we report the design of the first trial to use ONC201 as post-transplant maintenance (ClinicalTrials.gov ID: NCT03932643). Study Design and Methods: The study will follow a 3+3 phase 1 trial design. The primary objective is to determine the rate of dose-limiting toxicities (DLT) during the first cycle and grade ≥3 toxicities during the first 3 cycles. Key inclusion criteria include adult recipients of allogeneic hematopoietic stem cell transplant within 6-20 weeks for a history of high-risk AML or MDS; <5% bone marrow blasts; Karnofsky Performance Status of ≥70; absolute neutrophil count (ANC) greater than 1000/μL, and platelet count ³50,000/µL. Key exclusion criteria include a history of acute graft-versus-host disease (GVHD) grade III/IV; initiation of any new systemic immunosuppressive agent for GVHD within 4 weeks of enrollment, current use of prednisone at a dose of ≥0.25 mg/kg/day; uncontrolled serious infection; active ischemic heart disease, heart failure or arrhythmia; severe chronic obstructive pulmonary disease; resting O2 saturation <90%; aspartate transaminase, alanine transaminase or bilirubin >2 times the upper limit of normal; and creatinine clearance <30 mL/min. Patients will receive oral weekly ONC201 at a starting dose of 250 mg and dose escalation by 125 mg up to a maximum dose of 625 mg weekly, in the absence of DLT. Patients will be monitored for toxicities (using Common Terminology Criteria for Adverse Events, CTCAE version 5.0), quality of life (QOL) (Functional Assessment of Cancer Therapy-Bone Marrow Transplant, FACT-BMT), changes in functional status (KPS, instrumental activities of daily living and short physical performance battery), rates of disease relapse and mortality (See Study Schema). We will enroll a total of 20 patients; the first 6-9 patients are expected to receive escalating doses of ONC 201, and the remaining patients will receive the maximum tolerated doses. Clinical studies in solid malignancies have indicated that ONC201 can expand and activate NK cells in peripheral blood, or recruit CD8+ T-cells to the tumor. Hence, the study will assess expansion and activation of NK and T cell populations in peripheral blood and utilize transcriptomic analyses of NK and T cells to explore the mechanisms behind the immunologic changes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Identification and interrogation of the gene regulatory network of CEBPA-double mutant acute myeloid leukemia

Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy caused by mutations in genes encoding transcriptional and epigenetic regulators together with signaling genes. It is characterized by a disturbance of differentiation and abnormal proliferation of hematopoietic progenitors. We have previously shown that each AML subtype establishes its own core gene regulatory network (GRN), consisting of transcription factors binding to their target genes and imposing a specific gene expression pattern that is required for AML maintenance. In this study, we integrate gene expression, open chromatin and ChIP data with promoter-capture Hi-C data to define a refined core GRN common to all patients with CEBPA-double mutant (CEBPAN/C) AML. These mutations disrupt the structure of a major regulator of myelopoiesis. We identify the binding sites of mutated C/EBPα proteins in primary cells, we show that C/EBPα, AP-1 factors and RUNX1 colocalize and are required for AML maintenance, and we employ single cell experiments to link important network nodes to the specific differentiation trajectory from leukemic stem to blast cells. Taken together, our study provides an important resource which predicts the specific therapeutic vulnerabilities of this AML subtype in human cells.

HSF1 is a driver of leukemia stem cell self-renewal in acute myeloid leukemia

Acute myeloid leukemia (AML) is maintained by self-renewing leukemic stem cells (LSCs). A fundamental problem in treating AML is that conventional therapy fails to eliminate LSCs, which can reinitiate leukemia. Heat shock transcription factor 1 (HSF1), a central regulator of the stress response, has emerged as an important target in cancer therapy. Using genetic Hsf1 deletion and a direct HSF1 small molecule inhibitor, we show that HSF1 is specifically required for the maintenance of AML, while sparing steady-state and stressed hematopoiesis. Mechanistically, deletion of Hsf1 dysregulates multifaceted genes involved in LSC stemness and suppresses mitochondrial oxidative phosphorylation through downregulation of succinate dehydrogenase C (SDHC), a direct HSF1 target. Forced expression of SDHC largely restores the Hsf1 ablation-induced AML developmental defect. Importantly, the growth and engraftment of human AML cells are suppressed by HSF1 inhibition. Our data provide a rationale for developing efficacious small molecules to specifically target HSF1 in AML.

Inducement of ER Stress by PAD Inhibitor BB-Cl-Amidine to Effectively Kill AML Cells.

Acute myeloid leukemia (AML) is a highly heterogeneous and recurrent hematological malignancy. Despite the emergence of novel chemotherapy drugs, AML patients' complete remission (CR) remains unsatisfactory. Consequently, it is imperative to discover new therapeutic targets or medications to treat AML. Such epigenetic changes like DNA methylation and histone modification play vital roles in AML. Peptidylarginine deminase (PAD) is a protein family of histone demethylases, among which the PAD2 and PAD4 expression have been demonstrated to be elevated in AML patients, thus suggesting a potential role of PADs in the development or maintenance of AML and the potential for the identification of novel therapeutic targets. AML cells were treated in vitro with the pan-PAD inhibitor BB-Cl-Amidine (BB-Cl-A). The AML cell lines were effectively induced into apoptosis by BB-Cl-A. However, the PAD4-specific inhibitor GSK484 did not. PAD2 played a significant role in AML. Furthermore, we found that BB-Cl-A could activate the endoplasmic reticulum (ER) stress response, as evidenced by an increase in phosphorylated PERK (p-PERK) and eIF2α (p-eIF2α). As a result of the ER stress activation, the BB-Cl-A effectively induced apoptosis in the AML cells. Our findings indicated that PAD2 plays a role in ER homeostasis maintenance and apoptosis prevention. Therefore, targeting PAD2 with BB-Cl-A could represent a novel therapeutic strategy for treating AML.

Leukotrienes promote stem cell self-renewal and chemoresistance in acute myeloid leukemia.

Acute myeloid leukemia (AML) is characterized by poor clinical outcomes due to high rates of relapse following standard-of-care induction chemotherapy. While many pathogenic drivers have been described in AML, our understanding of the molecular mechanisms mediating chemotherapy resistance remains poor. Therefore, we sought to identify resistance genes to induction therapy in AML and elucidated ALOX5 as a novel mediator of resistance to anthracycline-based therapy. ALOX5 is transcriptionally upregulated in AML patient blasts in comparison to normal hematopoietic stem/progenitor cells (HSPCs) and ALOX5 mRNA, and protein expression is increased in response to induction therapy. In vitro, and in vivo genetic, and pharmacologic perturbation studies confirm that ALOX5 positively regulates the leukemogenic potential of AML LSCs, and its loss does not significantly affect the function of normal HSPCs. ALOX5 mediates resistance to daunorubicin (DNR) and promotes AML cell survival and maintenance through its leukotriene (LT) synthetic capacity, specifically via modulating the synthesis of LTB4 and its binding to LTB receptor (BLTR). Our study reveals a previously unrecognized role of LTs in AML pathogenesis and chemoresistance, whereby inhibition of ALOX5 mediated LTB4 synthesis and function could be combined with standard chemotherapy, to enhance the overall therapeutic efficacy in AML.

3221 – INVESTIGATING THE ROLE OF UBIQUITIN E3 LIGASE, FBXO21, IN THE REGULATION OF CYTOKINE SIGNALING WITHIN ACUTE MYELOID LEUKEMIA

Hematopoiesis is tightly regulated through various molecular mechanisms, including the ubiquitin proteasome system (UPS). Dysregulation can lead to malignancies, including acute myeloid leukemia (AML). AML is characterized by an abnormal proliferation of immature myeloid cells. F-box ubiquitin E3 ligase, FBXO21, is differentially expressed in both normal and malignant hematopoiesis. FBXO21 has low expression in AML, but high expression correlates to decreased survival in AML patients. At the protein level, AML patients with high blast counts have increased expression of FBXO21, suggesting FBXO21 may play a role in leukemic stem cells (LSCs) to promote progression and survival of AML. This paradoxical relationship between FBXO21 expression and patient survival highlights a major gap in knowledge and the necessity of further studies to understand the role of FBXO21 in AML. We have found that silencing of FBXO21 in AML cell lines and primary AML patient samples leads to increased apoptosis, differentiation, and decreased proliferation. Furthermore, silencing of FBXO21 leads to up-regulation of inflammatory cytokines/chemokines and modulation of MAP kinase pathways, which are known to be involved in the activation of many cytokine signaling pathways. We have identified up-regulation of CXCL10 at both the RNA and protein level, which is known to initiate T-cell recruitment and promote anti-tumor activity within the AML microenvironment. These findings lead us to hypothesize that FBXO21 regulates the maintenance and progression of AML through MAP kinase mediated cytokine signaling pathways. Regulation of cytokines and chemokines play a key role in the progression and maintenance of AML, suggesting FBXO21 may impact disease progression and survival.