Therapy Resistance In Acute Myeloid Leukemia Research Articles

BRD4 degraders may effectively counteract therapeutic resistance of leukemic stem cells in AML and ALL.

Acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) are life-threatening hematopoietic malignancies characterized by clonal expansion of leukemic blasts in the bone marrow and peripheral blood. The epigenetic reader BRD4 and its downstream effector MYC have recently been identified as potential drug targets in human AML and ALL. We compared anti-leukemic efficacies of the small-molecule BET inhibitor JQ1 and the recently developed BRD4 degraders dBET1 and dBET6 in AML and ALL cells. JQ1, dBET1, and dBET6 were found to suppress growth and viability in all AML and ALL cell lines examined as well as in primary patient-derived AML and ALL cells, including CD34+/CD38- and CD34+/CD38+ leukemic stem and progenitor cells, independent of the type (variant) of leukemia or molecular driver expressed in leukemic cells. Moreover, we found that dBET6 overcomes osteoblast-induced drug resistance in AML and ALL cells, regardless of the type of leukemia or the drug applied. Most promising cooperative or even synergistic drug combination effects were seen with dBET6 and the FLT3 ITD blocker gilteritinib in FLT3 ITD-mutated AML cells, and with dBET6 and the multi-kinase blocker ponatinib in BCR::ABL1+ ALL cells. Finally, all BRD4-targeting drugs suppressed interferon-gamma- and tumor necrosis factor-alpha-induced expression of the resistance-related checkpoint antigen PD-L1 in AML and ALL cells, including LSC. In all assays examined, the BRD4 degrader dBET6 was a superior anti-leukemic drug compared with dBET1 and JQ1. Together, BRD4 degraders may provide enhanced inhibition of multiple mechanisms of therapy resistance in AML and ALL.

Abstract 5592: HMGA1 modulates chromatin state and transcriptional networks involved in plasticity in refractory myeloid leukemia

Abstract Although High Mobility Group A1 (HMGA1) chromatin regulators are overexpressed in refractory acute myeloid leukemia (AML) and diverse solid tumors, targetable mechanisms underlying HMGA1 remain elusive. HMGA1 is highly expressed in stem cells and poorly differentiated cancers where it modulates chromatin structure to activate developmental gene networks. In AML arising from JAK2V617F myeloproliferative neoplasms (MPN), HMGA1 is required for leukemogenesis by inducing transcriptional networks that drive aberrant proliferation and cell fate. Like MPN AML, KMT2A-rearranged (KMT2A-r) AML is refractory to therapy and highly lethal. KMT2A-r AML is caused by rearrangements of the KMT2A gene, which encode abnormal fusion proteins that induce pro-leukemogenic genes, including HOX genes. While prior work identified KMT2A fusion partners and protein complexes, this has not led to better therapies for most patients. We therefore sought to examine HMGA1 function in KMT2A-r AML. We hypothesized that: 1) HMGA1 drives plasticity in KMT2A-r AML by altering chromatin state and transcriptional networks, and, 2) Targeting HMGA1 will enhance sensitivity to therapy. In two KMT2A-r AML patient cohorts, HMGA1 is highly overexpressed compared to CD34+ stem and progenitors from healthy controls, suggesting it drives leukemogenesis. To define HMGA1 function, we silenced HMGA1 using CRISPR or short hairpin RNA in KMT2A-r cell lines (MOLM-14, THP-1, MV4-11). Strikingly, silencing HMGA1 disrupts proliferation and clonogenicity in vitro, while prolonging survival in immunosuppressed mice transplanted with KMT2A-r AML cells. Intriguingly, leukemic cells that engraft in the bone marrow from the pool of cells with HMGA1 silencing express higher levels of HMGA1, suggesting that escape from gene silencing and a specific level of HMGA1 is required for leukemic engraftment and expansion. HMGA1 also increases in KMT2A-r AML cells that become resistant to cytarabine (AraC), suggesting that HMGA1 endows AML cells with the capacity to survive and expand after AraC. Because repression of tumor suppressors was recently defined as a mechanism of therapy resistance in AML, we also examined tumor suppressor pathways in both JAK2V617F and KMT2A-r AML. Intriguingly, HMGA1 occupies the promoter region of the CDKN1A locus by chromatin immunoprecipitation and represses its expression in KMT2A-r and JAK2V617F AML cells. To define additional mechanisms underlying HMGA1 in KMT2A-r AML, multi-omics studies are underway. In preliminary work thus far, we also found that HMGA1 induces HOX genes. Together, these findings implicate HMGA1 as a promising therapeutic target and novel epigenetic regulator in KMT2A-r AML by altering chromatin state and downstream gene expression. Citation Format: Bailey E. West, Jung-Hyun Kim, Audrey-Ann Supreme, Iliana Herrera, Zanshé Thompson, Li Z. Luo, Faiza Shaik, Joseph Kim, Hyunsung Woo, Soheil Meshinchi, Rhonda E. Ries, Linda M. Resar. HMGA1 modulates chromatin state and transcriptional networks involved in plasticity in refractory myeloid leukemia [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 5592.

Evaluation of apoptosis stimulating protein of TP53-1 (ASPP1/PPP1R13B) to predict therapy resistance and overall survival in acute myeloid leukemia (AML)

ASPP1 (PPP1R13B) belongs to a family of p53-binding proteins and enhances apoptosis by stimulation of p53-transactivation of selected proapoptotic target genes. It is preferentially expressed in hematopoietic stem cells (HSC) and together with p53 preserves the genomic integrity of the HSC pool. Consequently, dysfunction of ASPP1 has been associated with malignant transformation and development of acute lymphoblastic leukemias and lymphomas - whereas methylation of the promoter region is linked to reduced transcription and ultimately attenuated expression of ASPP1. The role of ASPP1 in AML is not known. We now show that impaired regulation of PPP1R13B contributes to the biology of leukemogenesis and primary therapy resistance in AML. PPP1R13B mRNA expression patterns thereby define a distinct prognostic profile - which is not reflected by the European leukemia net (ELN) risk score. These findings have direct therapeutic implications and we provide a strategy to restore ASPP1 protein levels using hypomethylating agents to sensitize cells towards proapoptotic drugs. Prospective clinical trials are warranted to investigate the role of ASPP1 (PPP1R13B) as a biomarker for risk stratification and as a potential therapeutic target to restore susceptibility to chemotherapy.

Aberrant Lipid Metabolic Signatures in Acute Myeloid Leukemia.

Leukemogenesis is a complex process that involves multiple stages of mutation in either hematopoietic stem or progenitor cells, leading to cancer development over time. Acute myeloid leukemia (AML) is an aggressive malignancy that affects myeloid cells. The major disease burden is caused by immature blast cells, which are eliminated using conventional chemotherapies. Unfortunately, relapse is a leading cause of death in AML patients, with 30%-80% experiencing it within 2 years of initial treatment. The dominant cause of relapse in leukemia is the presence of therapy-resistant leukemic stem cells (LSCs). These cells express genes related to stemness that are frequently difficult to eradicate and tend to survive standard treatments. Studies have demonstrated that by targeting the metabolic pathways of LSCs, it is possible to improve outcomes and extend the survival of those afflicted by leukemia. The overwhelming evidence suggests that lipid metabolism is reprogrammed in LSCs, leading to an increase in fatty acid uptake and de novo lipogenesis. Genes regulating this process also play a crucial role in therapy evasion. In this concise review, we summarize the lipid metabolism in normal hematopoietic cells, AML blast cells, and AML LSCs. We also compare the lipid metabolic signatures in de novo versus therapy-resistant AML blast and LSCs. We further discuss the metabolic switches, cellular crosstalk, potential targets, and inhibitors of lipid metabolism that could alleviate treatment resistance and relapse.

Phosphoproteomic Characterization and Kinase Signature Predict Response to Venetoclax Plus 3+7 Chemotherapy in Acute Myeloid Leukemia.

Resistance to chemotherapy remains a formidable obstacle in acute myeloid leukemia (AML) therapeutic management, necessitating the exploration of optimal strategies to maximize therapeutic benefits. Venetoclax with 3+7 daunorubicin and cytarabine (DAV regimen) in young adult de novo AML patients is evaluated. 90% of treated patients achieved complete remission, underscoring the potential of this regimen as a compelling therapeutic intervention. To elucidate underlying mechanisms governing response to DAV in AML, quantitative phosphoproteomics to discern distinct molecular signatures characterizing a subset of DAV-sensitive patients is used. Cluster analysis reveals an enrichment of phosphoproteins implicated in chromatin organization and RNA processing within DAV-susceptible and DA-resistant AML patients. Furthermore, kinase activity profiling identifies AURKB as a candidate indicator of DAV regimen efficacy in DA-resistant AML due to AURKB activation. Intriguingly, AML cells overexpressing AURKB exhibit attenuated MCL-1 expression, rendering them receptive to DAV treatment and maintaining them resistant to DA treatment. Moreover, the dataset delineates a shared kinase, AKT1, associated with DAV response. Notably, AKT1 inhibition augments the antileukemic efficacy of DAV treatment in AML. Overall, this phosphoproteomic study identifies the role of AURKB as a predictive biomarker for DA, but not DAV, resistance and proposes a promising strategy to counteract therapy resistance in AML.

Increased Monocytic Myeloid-Derived Suppressor Cells Are Associated with Inferior Overall Survival in Patients with Newly Diagnosed Acute Myeloid Leukemia

Background: Acute myeloid leukemia (AML) is an aggressive malignancy associated with poor long-term outcomes. This malignancy arises in the context of an immunosuppressive milieu, which fosters immune escape and tumor growth. Myeloid-derived suppressor cells (MDSCs) represent a heterogeneous group of immature myeloid cells with immunosuppressive activity, the most potent of which are the monocytic MDSCs (mMDSCs). The impact of mMDSCs on AML relapse and overall survival has yet to be fully characterized. Therefore, we sought to address this unanswered question through a retrospective analysis of a cohort of AML patients (pts) at Roswell Park Comprehensive Cancer Center. Methods: Medical records were retrospectively reviewed under an IRB approved protocol to identify pts aged ≥ 18 years old with newly diagnosed AML treated with standard cytarabine and anthracycline based chemotherapy from 2004-2017 at Roswell Park Comprehensive Cancer Center. Demographics, disease-specific variables, baseline clinical characteristics, and treatment response were analyzed using descriptive statistics. Detailed analysis of previously collected clinical multiparameter flow cytometric data was performed utilizing WinList software to identify mMDSCs at serial clinical time points (diagnosis, after induction chemotherapy, and relapse). A mononuclear gate was created utilizing CD45 vs. SSC (blasts excluded), followed by FSC vs. SSC to eliminate dead cells and aggregates. mMDSCs were defined as the subset of marrow cells co-expressing CD14+ and HLA-DR dim and was reported as the percentage of total monocytes in the marrow aspirate sample. mMDSC populations were dichotomized into “Low” and “High” groups based on the median value of the entire cohort. Overall survival (OS) and relapse-free survival (RFS) were estimated utilizing Kaplan-Meier (KM) analysis. Results: 81 pts with newly diagnosed AML who received induction with a cytarabine and anthracycline based regimen were identified. Median age was 55 years (range 20 - 85), with 52 men (64.2%) (Table 1). Cytogenetics per ELN 2022 were high risk in 38 pts (46.9%), intermediate risk in 28 pts (34.6%), and favorable risk in 15 pts (18.5%). 6/69 (8.7%) pts had FLT3-ITD mutations. Following induction therapy, 15 pts (18.5%) had primary refractory disease with 66 (81.5%) achieving complete remission (CR). 28 pts (34.6%) underwent allo HSCT. Pts with intermediate risk disease and “High” mMDSCs at time of diagnosis and post induction chemotherapy had inferior OS compared to those with “Low” mMDSCs (16 vs. 34 months, P=0.008; 16 vs. 27 months, P=0.03) (Figure 1). There were no significant differences in RFS in intermediate or poor risk pts, and no difference in OS in poor risk pts when comparing “Low” vs. “High” mMDSCs at any time point. Mean duration of follow up was 23.7 months (range 1 - 148 months). Analysis of favorable risk cohort is ongoing. Conclusions: This retrospective analysis suggests that high numbers of marrow mMDSCs are associated with poor overall survival in pts with intermediate risk AML receiving intensive induction therapy. Of note, other risk factors for poor risk disease such as complete molecular profiling were not available. However, our findings support a potential role of mMDSCs in promoting disease recurrence. Additional studies to further quantify and delineate the biological role of mMDSCs in a larger pt cohort are needed to corroborate these findings and determine the potential role of these immune cells in therapy resistance in AML.

Identifying Bone Marrow Microenvironmental Populations in Myelodysplastic Syndrome and Acute Myeloid Leukemia.

The bone marrow microenvironment consists of distinct cell populations, such as mesenchymal stromal cells, endothelial cells, osteolineage cells, and fibroblasts, which provide support for hematopoietic stem cells (HSCs). In addition to supporting normal HSCs, the bone marrow microenvironment also plays a role in the development of hematopoietic stem cell disorders, such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). MDS-associated mutations in HSCs lead to a block in differentiation and progressive bone marrow failure, especially in the elderly. MDS can often progress to therapy-resistant AML, a disease characterized by a rapid accumulation of immature myeloid blasts. The bone marrow microenvironment is known to be altered in patients with these myeloid neoplasms. Here, a comprehensive protocol to isolate and phenotypically characterize bone marrow microenvironmental cells from murine models of myelodysplastic syndrome and acute myeloid leukemia is described. Isolating and characterizing changes in the bone marrow niche populations can help determine their role in disease initiation and progression and may lead to the development of novel therapeutics targeting cancer-promoting alterations in the bone marrow stromal populations.

The relevance of the hematopoietic niche for therapy resistance in acute myeloid leukemia.

The expansion of acute myeloid leukemia (AML) blasts not only suppresses normal hematopoiesis, but also alters the microenvironment. The interplay of different components of the bone marrow gives rise to altered metabolic states and activates signaling pathways which lead to resistance and impede effective therapy. Therefore, the underlying processes and mechanisms represent attractive therapeutic leverage points for overcoming therapy resistance in AML. Here, we briefly discuss resistance mechanisms based on cell interactions and secreted soluble factors in the hematopoietic niche and provide an overview of niche-related therapeutic targets currently undergoing preclinical and clinical investigation which may help improve the outcome in AML therapy.

Single-cell chromatin accessibility profiling of acute myeloid leukemia reveals heterogeneous lineage composition upon therapy-resistance

Acute myeloid leukemia (AML) is a heterogeneous disease characterized by high rate of therapy resistance. Since the cell of origin can impact response to therapy, it is crucial to understand the lineage composition of AML cells at time of therapy resistance. Here we leverage single-cell chromatin accessibility profiling of 22 AML bone marrow aspirates from eight patients at time of therapy resistance and following subsequent therapy to characterize their lineage landscape. Our findings reveal a complex lineage architecture of therapy-resistant AML cells that are primed for stem and progenitor lineages and spanning quiescent, activated and late stem cell/progenitor states. Remarkably, therapy-resistant AML cells are also composed of cells primed for differentiated myeloid, erythroid and even lymphoid lineages. The heterogeneous lineage composition persists following subsequent therapy, with early progenitor-driven features marking unfavorable prognosis in The Cancer Genome Atlas AML cohort. Pseudotime analysis further confirms the vast degree of heterogeneity driven by the dynamic changes in chromatin accessibility. Our findings suggest that therapy-resistant AML cells are characterized not only by stem and progenitor states, but also by a continuum of differentiated cellular lineages. The heterogeneity in lineages likely contributes to their therapy resistance by harboring different degrees of lineage-specific susceptibilities to therapy.

C/EBPα Confers Dependence to Fatty Acid Biosynthetic Pathways and Vulnerability to Lipid Oxidative Stress in FLT3-Mutant Leukemia

Despite therapeutic breakthroughs, drug resistance and relapse remain the major barrier for the treatment of patients with acute myeloid leukemia (AML). Therefore, understanding and targeting therapy-resistant AML cells responsible for relapse ("persisters") represent an urgent need in the development of new treatments. In this context, we demonstrated that the hyperactivation of mitochondrial oxidative metabolism plays a crucial role in drug resistance of AML. This high oxidative phosphorylation (OxPHOS) phenotype associated with enhanced mitochondrial BCL2 dependency and ROS detoxification is the consequence of a mitochondrial adaptation driven by inflammatory stress responses in a cAMP-PKA-ATF4-dependent manner. Targeting elevated oxidative phosphorylation (OxPHOS) activity with direct and indirect mitochondrial inhibitors sensitizes resistant cells to chemotherapy cytarabine (AraC) in AML. Furthermore, we have recently shown that the mitochondrial OxPHOS state supports IDH mutant cell proliferation and chemoresistance in a C/EBPa-dependent manner. Importantly, multi-omics analyses uncovered a coordinated activation of CEBPA and Fms-like tyrosine kinase 3 (FLT3) to control lipid metabolism in FLT3-mutant AML cells from primary AML patient specimens, cell lines, and PDX models. Mechanistically, C/EBPα regulated FASN-SCD axis to promote uptake, biosynthesis and desaturation of fatty acids (FA). We further demonstrated that FLT3 or C/EBPα inactivation decreased mono-unsaturated FAs incorporation to membrane phospholipids through SCD downregulation in leukemic cells. Consequently, SCD inhibition enhanced susceptibility to lipid redox stress and accumulation of poly-unsaturated FA increased lipid peroxidation and vulnerability to redox stress. Accordingly, this C/EBPα-dependent adaptation of FA homeostasis was exploited by combining FLT3 and glutathione peroxidase 4 (GPX4) inhibition to trigger lipid oxidative stress, enhancing ferroptotic death of FLT3-mutant AML cells ex vivo and in vivo. Altogether, this study reveals a non-canonical C/EBPα function in lipid homeostasis and adaptation to redox stress, and a previously unreported vulnerability of FLT3-mutant AML with promising therapeutic application. Altogether, our works highlight the role of metabolic adaptations to support the plasticity and drug resistance in AML.

Microenvironment and drug resistance in acute myeloid leukemia: Do we know enough?

Acute myeloid leukemia (AMLs), as the name suggests, often develop suddenly and are very progressive forms of cancer. Unlike in acute promyelocytic leukemia, a subtype of AML, the outcomes in most other AMLs remain poor. This is mainly attributed to the acquired drug resistance and lack of targeted therapy. Different studies across laboratories suggest that the cellular mechanisms to impart therapy resistance are often very dynamic and should be identified in a context-specific manner. Our review highlights the progress made so far in identifying the different cellular mechanisms of mutation-independent therapy resistance in AML. It reiterates that for more effective outcomes cancer therapies should acquire a more tailored approach where the protective interactions between the cancer cells and their niches are identified and targeted.

Leukemia Stem Cells Depend on Regulated Protein Synthesis

Acute myeloid leukemia (AML) is initiated and sustained by leukemia stem cells (LSCs) which arise from progenitor cells that do not usually self-renew but become aberrantly self-renewing. It is thought that LSCs gain aberrant self-renewal potential by co-opting molecular and cellular programs from hematopoietic stem cells (HSCs) (PMID: 16862118). HSCs have been shown to require tightly regulated protein synthesis rates, where increased or decreased protein synthesis impairs self-renewal (PMID: 24670665), but it is not known if LSCs share this dependence. We have shown that human LSCs reside in the population of AML cells with the highest levels of CD99 (PMID: 28123069). In RNA-sequencing studies, we found that LSCs with high levels of CD99 are depleted for ribosomal protein transcripts. We thus reasoned that similar to HSCs, LSCs may depend on tightly regulated protein synthesis to self-renew.To test if CD99 promotes LSC function by constraining protein synthesis, we transduced c-Kit+ cells from B6-CD99 Gt(pU-21T)44lmeg (CD99 KO) or wild-type (WT) mice to express AML1-ETO9a (AE9a) and transplanted them into WT mice treated with rapamycin or vehicle. There was no difference in leukemogenesis in primary recipients, but CD99 KO-AE9a AMLs exhibited a 72% (p=0.048) increase in protein synthesis compared with WT-AE9a AMLs (Figure 1A), confirming that CD99 negatively regulates protein synthesis in AML. We next performed secondary transplants to assess LSC function, as measured by survival of secondary recipients in the absence of rapamycin treatment (Figure 1B). We furthermore performed these transplants at limiting dilution to quantify LSCs (Figure 1C). CD99 KO-AE9a vehicle treated AMLs demonstrated improved survival and a lower LSC frequency compared with WT-AE9a vehicle treated AMLs, consistent with a self-renewal defect with loss of CD99. Rapamycin treatment completely rescued this defect, leading to decreased survival and increased LSC frequency in CD99 KO-AE9a AMLs compared with vehicle. Conversely, rapamycin treatment depleted LSCs in WT-AE9a AMLs, increasing survival and decreasing LSC frequency compared with vehicle. Thus, similar to HSCs, LSCs are adversely affected by both increases or decreases in protein synthesis.MLL-AF9-induced mouse AMLs initiated in HSCs as compared with granulocyte macrophage progenitors (GMPs) exhibit increased epigenetic imprinting of HSC features resulting in disease features reminiscent of high-risk AML (PMID: 23235717). To test if HSC-derived leukemias exhibit increased dependence on regulated protein synthesis, we transduced HSCs or GMPs from CD99 KO or WT mice to express MLL-AF9 and transplanted them into WT recipients, followed by secondary transplants to assess LSC function. Loss of CD99 led to increased survival indicative of decreased LSC function in HSC-derived but not GMP-derived leukemias (Figure 1D). This suggests that HSC-derived leukemias co-opt from HSCs a more pronounced dependence on tightly regulated protein synthesis. Accordingly, WT HSC-derived leukemias exhibited decreased protein synthesis as compared with their WT GMP-derived counterparts (Figure 1E), as well as increased sensitivity to rapamycin (Figure 1F).To directly study protein synthesis in human LSCs, we transduced primary AML specimens (n=4) to express a destabilized form of GFP (dGFP) from a constitutive promoter followed by xenotransplantation (Figure 1G), allowing us to measure dGFP by flow cytometry as a surrogate for protein synthesis rates in vivo. We validated this assay by measuring protein synthesis using orthogonal O-propargyl-puromycin incorporation assays (Figure 1H). Human AML cells with low levels of dGFP demonstrated increased engraftment in secondary transplants (Figure 1I), demonstrating that human LSCs exhibit low protein synthesis rates.In conclusion, our data demonstrate that LSCs co-opt from HSCs a dependence on tightly regulated protein synthesis. This is the first description of a cellular feature co-opted from HSCs that also represents a therapeutic vulnerability. Furthermore, the types of AML that exhibit the most robust re-activation of HSC programs and increased dependence on regulated protein synthesis are also likely to represent high-risk AMLs most resistant to standard therapies. Our data suggest that such therapy resistant AMLs may be highly sensitive to strategies disrupting protein synthesis to deplete LSCs. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Mannose Metabolism Is a Metabolic Vulnerability Unveiled By Standard and Novel Therapies in Acute Myeloid Leukemia

IntroductionThe development of resistance to standard and novel therapies remains the main obstacle to cure in acute myeloid leukemia (AML). Metabolic rewiring has emerged as a therapeutically actionable vulnerability in AML, where specific metabolic adaptations arising as a result of driver mutations or in response to therapy have been reported. Mannose phosphoisomerase (MPI) is the first enzyme in the mannose metabolism (MM) pathway, that leads to the production of GDP-Mannose, a key sugar donor for N-glycosylation reactions. MPI was amongst the top drop-out genes in our published CRISPR-Cas9 screen aiming to identify sensitizers to FLT3 tyrosine kinase inhibitors (TKI) in AML carrying activating FLT3 internal tandem duplication (ITD) mutations (Gallipoli et al., Blood 2018). Moreover, analysis of published genomic datasets indicates that MPI expression levels are higher in AML compared to normal samples, correlate with patient outcome (A) and further increase in paired relapsed to diagnosis samples. We therefore hypothesized that MM and MPI inhibition sensitize AML cells to both FLT3-TKI and standard chemotherapy and tested this in preclinical models.MethodsExperiments were performed in human AML cell lines, primary AML mononuclear cells and normal CD34 + stem/progenitor cells. Liquid chromatography coupled to mass spectrometry and oxygen consumption/extracellular acidification rate as measured by a Seahorse analyser were employed to assess metabolic changes. Gene-expression was measured by RNA-sequencing and confirmed by RT-qPCR. Viability, surface protein expression and lipid peroxidation were assessed by flow-cytometry. Protein expression and localisation was measured by western blot and immunofluorescence. Gene silencing was performed using CRISPR-Cas9 gene editing and inducible short hairpin RNA interference.ResultsWe show that genetic and chemical MPI inhibition sensitizes FLT3 WT and FLT3 ITD AML cell lines in vitro to standard cytarabine chemotherapy and FLT3-TKI respectively and these effects are rescued by the addition of MPI downstream product mannose. We validate these findings in vivo using MPI knock-out (KO) cell line xenografts and in vitro using primary AML samples following MPI knock-down (B). We also demonstrate a lack of toxicity to normal CD34 + cells.Surprisingly, global metabolomic analysis show that MPI KO cells accumulate fatty acids and particularly polyunsaturated fatty acids (PUFA) (C), due to both increased uptake and reduced fatty acid oxidation (FAO). MPI KO cells downregulate several FAO metabolism genes and this is corroborated by the strong positive correlation of MPI expression levels with multiple genes involved in FAO across multiple primary AML datasets. Metabolic profiling demonstrates that MPI KO cells have reduced oxidative phosphorylation metabolism and in particular are unable to oxidise palmitate, an effect rescued by mannose or the FAO activator fenofibrate (D).We link this metabolic defect to the specific activation of the ATF6 arm of the unfolded protein response (UPR) due to a reduction in protein glycosylation in MPI KO cells. We validate this by demonstrating that activating or inhibiting ATF6 respectively phenocopies or rescues the effects of MPI KO (E) and show this to be secondary to ATF6 driving FAO inhibition, via transcriptional downregulation of PPARα, a master regulator of lipid catabolism.Finally, we show that accumulation of PUFA in MPI KO cells is accompanied by reduced cysteine levels and increased markers of oxidative stress and lipid peroxidation, such as 4-hydroxynonenal (4-HNE), all features associated with ferroptosis (F). We validate this by showing that combining FLT3-TKI with MPI KO leads to ferroptotic cell death in AML cells, which can be rescued by the radical-trapping antioxidant ferrostatin.ConclusionsWe provide further evidence to support the role of metabolic rewiring in driving therapy resistance in AML and show for the first time that targeting MPI and MM sensitizes AML cells to cytarabine and FLT3-TKI. Mechanistically we unveil a novel connection between MM and fatty acid metabolism, via activation of the ATF6 arm of the UPR, leading to cellular PUFA accumulation, lipid peroxidation and ferroptotic cell death. Finally, our findings also suggest that triggering ferroptosis can be leveraged as a therapeutically actionable mechanism driving cell death in therapy-resistant AML cells. [Display omitted] DisclosuresVassiliou: Kymab Ltd: Divested equity in a private or publicly-traded company in the past 24 months; STRM.BIO: Consultancy; Astrazeneca: Consultancy.

Co-Targeting E-Selectin/CXCR4 with GMI-1359 Facilitates AML Stem Cell Mobilization and Protects BM Niches from Anti-Leukemia Therapy

Acute myeloid leukemia (AML) is characterized by the heterogeneous clonal expansion of undifferentiated myeloid cells in the bone marrow (BM). AML cells compete with normal hematopoietic cells and rewire the BM microenvironment into niches that selectively support leukemia stem cells (LSC). The leukemic niche produces soluble factors that facilitate the retention of LSC and provide protection from cytotoxic and targeted agents. The vascular adhesion molecule, E-selectin is expressed on endothelial cells (EC) in the perivascular niche where therapy-resistant AML cells have an increased affinity to E-selectin compared to normal hematopoietic stem cells (HSC) (Winkler et al., 2020). We previously demonstrated (Chang et al., ASH 2020) that E-selectin blockade by the pharmacological antagonist, GMI-1271 (uproleselan; GlycoMimetics, Inc) sensitized therapy-resistant LSC to Bcl-2 targeted therapy. Efficacious eradication of LSC in the BM however requires blocking multiple receptors and/or associated signaling pathways. A more optimal dislodgement of LSC from the BM could be attained by combining an E-selectin antagonism with blockade of the CXCR4/SDF-1α axis. The dual antagonist of E-selectin and CXCR4, GMI-1359 (GlycoMimetics, Inc.), has been tested in a phase 1 clinical trial (NCT02931214). Previously, we showed that GMI-1359 in combination with a FLT3-ITD inhibitor, improved survival in a xenograft model of FLT3-ITD + AML (Zhang et al., 2016). Hence, we hypothesized that co-targeting E-selectin/CXCR4 more efficiently mobilizes AML cells from BM niches and synergizes with the anti-leukemia activity of venetoclax/hypomethylating agent (Ven/HMA).Intra-vital 2-photon imaging and tracking of individual leukemia cells in triple reporter mice (Blood: dextran-TRITC; Host T-cells: DsRed; Host myeloid CD11 cells: EYFP) injected with AML cells carrying a turquoise fluorescent protein reporter gene suggested that dual inhibition of E-selectin/CXCR4 with GMI-1359 significantly enhanced AML cell motility (Fig 1. from 2.2 um/min to 5.4 um/min, p<0.001). Individual cells were dislodged from the niche and traveled long-distance. The combined inhibition of E-selectin and CXCR4 depleted BM leukemia cells in vascular niche areas. In a patient-derived primary AML xenograft (PDX) model (harboring mutations in JAK2 and c-Kit), combinatorial treatment of GMI-1359 with Ven/HMA significantly reduced BM retention of LSC compared to control cohorts or to Ven/HMA alone (p = 0.02 and p=0.003, respectively).In order to better understand how the augmented AML mobilization improves the efficacy of AML therapy, BM cells from PDX mice treated for 2 weeks with GMI-1271, GMI-1359, Ven/HMA, and their combinations were analyzed by single-cell proteomics (CyTOF). Blockade of E-selectin alone or dual E-selectin/CXCR4 inhibition in combination with Ven/HMA diminished levels of E-selectin ligand, mTOR, pFAK, pRb, cMyc, while increasing p21 and cleaved caspase3, which was associated with significant reduction of BM-resident LSC compared to Ven/HMA alone (CD45+34+CD38-CD123+, p= 0.03). AML blasts from the BM of the combinatorial treatment groups showed altered signaling including decreased Ki67, pRb, pNFkB, pPI3K, and E-selectin ligand, and increased levels of cleaved caspase 3. We further found that Ven/HMA significantly diminished CD31+ EC in the BM compared to control cohorts (p= 0.009). However, pharmacological antagonists of E-selectin or E-selectin/CXCR4 protected EC from Ven/HMA-induced detrimental insults through upregulation of survival signaling cascades including pAKT, pERK, pMAPK and decreased eNOS expression in EC compared to Ven/HMA treatment alone. Both EC and MSC were protected by dual inhibition of E-selectin/CXCR4 with GMI-1359. We also observed upregulated pro-survival signaling pathways such as phosphorylation of AKT-MAPK-ERK along with increased Bcl-xL, Bcl-2, and Idu expression in MSC from the GMI-1359 + Ven/HMA treated PDX mice compared to Ven/HMA single treatment cohorts.Collectively, our results provide strong evidence that co-targeting E-selectin/CXCR4 with GMI-1359 profoundly reduces BM retention of LSC as well as protects BM niche component cells from apoptosis induced by targeted therapy, resulting in improving the anti-leukemia activity of Ven/HMA therapy in AML. [Display omitted] DisclosuresFogler: GlycoMimetics Inc.: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Magnani: GlycoMimetics Inc.: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Carter: Ascentage: Research Funding; Syndax: Research Funding. Andreeff: Oxford Biomedica UK: Research Funding; ONO Pharmaceuticals: Research Funding; AstraZeneca: Research Funding; Reata, Aptose, Eutropics, SentiBio; Chimerix, Oncolyze: Current holder of individual stocks in a privately-held company; Karyopharm: Research Funding; Breast Cancer Research Foundation: Research Funding; Syndax: Consultancy; Daiichi-Sankyo: Consultancy, Research Funding; Novartis, Cancer UK; Leukemia & Lymphoma Society (LLS), German Research Council; NCI-RDCRN (Rare Disease Clin Network), CLL Foundation; Novartis: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Aptose: Consultancy; Glycomimetics: Consultancy; Medicxi: Consultancy; Senti-Bio: Consultancy.

Long Non-Coding RNA LINC00152 Regulates Self-Renewal of Leukemia Stem Cells and Induces Chemo-Resistance in Acute Myeloid Leukemia.

Relapse of acute myeloid leukemia (AML) has a very poor prognosis and remains a common cause of treatment failure in patients with this disease. AML relapse is partially driven by the chemoresistant nature of leukemia stem cells (LSCs), which remains poorly understood, and our study aimed at elucidating the underlying mechanism. Accumulating evidences show that long noncoding RNAs (lncRNAs) play a crucial role in AML development. Herein, the lncRNA, LINC00152, was identified to be highly expressed in CD34+ LSCs and found to regulate the self-renewal of LSCs derived from AML patients. Importantly, LINC00152 upregulation was correlated with the expression of 16 genes within a 17-gene LSC biomarker panel, which contributed to the accurate prediction of initial therapy resistance in AML. Knockdown of LINC00152 markedly increased the drug sensitivity of leukemia cells. Furthermore, LINC00152 expression was found to be correlated with poly (ADP-ribose) polymerase 1 (PARP1) expression in AML, whereas LINC00152 knockdown significantly decreased the expression of PARP1. Upregulation of LINC00152 or PARP1 was associated with poor prognosis in AML patients. Collectively, these data highlight the importance and contribution of LINC00152 in the regulation of self-renewal and chemoresistance of LSCs in AML.

Inhibiting autophagy targets human leukemic stem cells and hypoxic AML blasts by disrupting mitochondrial homeostasis

AbstractLeukemia stem cells (LSCs) and therapy-resistant acute myeloid leukemia (AML) blasts contribute to the reinitiation of leukemia after remission, necessitating therapeutic interventions that target these populations. Autophagy is a prosurvival process that allows for cells to adapt to a variety of stressors. Blocking autophagy pharmacologically by using mechanistically distinct inhibitors induced apoptosis and prevented colony formation in primary human AML cells. The most effective inhibitor, bafilomycin A1 (Baf A1), also prevented the in vivo maintenance of AML LSCs in NSG mice. To understand why Baf A1 exerted the most dramatic effects on LSC survival, we evaluated mitochondrial function. Baf A1 reduced mitochondrial respiration and stabilized PTEN-induced kinase-1 (PINK-1), which initiates autophagy of mitochondria (mitophagy). Interestingly, with the autophagy inhibitor chloroquine, levels of enhanced cell death and reduced mitochondrial respiration phenocopied the effects of Baf A1 only when cultured in hypoxic conditions that mimic the marrow microenvironment (1% O2). This indicates that increased efficacy of autophagy inhibitors in inducing AML cell death can be achieved by concurrently inducing mitochondrial damage and mitophagy (pharmacologically or by hypoxic induction) and blocking mitochondrial degradation. In addition, prolonged exposure of AML cells to hypoxia induced autophagic flux and reduced chemosensitivity to cytarabine (Ara-C), which was reversed by autophagy inhibition. The combination of Ara-C with Baf A1 also decreased tumor burden in vivo. These findings demonstrate that autophagy is critical for mitochondrial homeostasis and survival of AML cells in hypoxia and support the development of autophagy inhibitors as novel therapeutic agents for AML.

Bone marrow stromal cells induce an ALDH+ stem cell-like phenotype and enhance therapy resistance in AML through a TGF-β-p38-ALDH2 pathway.

The bone marrow microenvironment (BME) in acute myeloid leukemia (AML) consists of various cell types that support the growth of AML cells and protect them from chemotherapy. Mesenchymal stromal cells (MSCs) in the BME have been shown to contribute immensely to leukemogenesis and chemotherapy resistance in AML cells. However, the mechanism of stroma-induced chemotherapy resistance is not known. Here, we hypothesized that stromal cells promote a stem-like phenotype in AML cells, thereby inducing tumorigenecity and therapy resistance. To test our hypothesis, we co-cultured AML cell lines and patient samples with BM-derived MSCs and determined aldehyde dehydrogenase (ALDH) activity and performed gene expression profiling by RNA sequencing. We found that the percentage of ALDH+ cells increased dramatically when AML cells were co-cultured with MSCs. However, among the 19 ALDH isoforms, ALDH2 and ALDH1L2 were the only two that were significantly upregulated in AML cells co-cultured with stromal cells compared to cells cultured alone. Mechanistic studies revealed that the transforming growth factor-β1 (TGF-β1)-regulated gene signature is activated in AML cells co-cultured with MSCs. Knockdown of TGF-β1 in BM-MSCs inhibited stroma-induced ALDH activity and ALDH2 expression in AML cells, whereas treatment with recombinant TGF-β1 induced the ALDH+ phenotype in AML cells. We also found that TGF-β1-induced ALDH2 expression in AML cells is mediated by the non-canonical pathway through the activation of p38. Interestingly, inhibition of ALDH2 with diadzin and CVT-10216 significantly inhibited MSC-induced ALDH activity in AML cells and sensitized them to chemotherapy, even in the presence of MSCs. Collectively, BM stroma induces ALDH2 activity in AML cells through the non-canonical TGF-β pathway. Inhibition of ALDH2 sensitizes AML cells to chemotherapy.

Imetelstat Inhibits Telomerase and Prevents Propagation of ADAR1-Activated Myeloproliferative Neoplasm and Leukemia Stem Cells

BACKGROUND Clonal stem cell derived myeloproliferative neoplasms (MPNs) have a propensity to evolve to acute myeloid leukemia (AML). Deregulation of the innate immune deaminase associated with RNA1 (ADAR1) has been linked to malignant progression and therapeutic resistance. Increased expression of the stem cell gene, human telomerase reverse transcriptase (hTERT), has also been linked with malignant transformation. However, the combinatorial role of ADAR1 and hTERT in the evolution of MPN stem cells to therapy resistant acute myeloid leukemia stem cells (LSCs) and the capacity of a telomerase inhibitor, imetelstat, to prevent survival and self-renewal of pre-LSC and LSC had not been established. Recent clinical trials show early signs of efficacy of imetelstat in treatment of myelofibrosis (MF). However, its role in selectively inhibiting pre-LSC transformation to self-renewing LSC has not been elucidated. Here we show that targeting telomerase activity prevents pre-LSC and LSC maintenance both in vitro and in vivo, suggesting telomerase inhibition as an effective strategy for preventing MPN progression. METHODS To quantify hTERT level and ADAR1 activity in the setting of normal HSPC and MPN stem cell evolution, whole genome sequencing (WGS) analysis was performed on 76 normal and MPN blood CD34+ cells and matching saliva samples. Results were compared with RNA-seq of 100 FACS purified young, aged, MPN and AML CD34+CD38- stem cells and CD34+CD38+ progenitor cells. Confocal fluorescence microscopic evaluation of stem cell ADAR1 and hTERT localization, telomere length by Flow-FISH and telomerase activity by TRAP assays, lentiviral ADAR1 overexpression and shRNA knockdown were performed. In vitro stromal co-cultures, and humanized immunocompromised mouse models were established to determine the impact of imetelstat (a oligonucleotide inhibitor of telomerase) on normal, MPN stem cell and LSC maintenance. RESULTS Combined hTERT overexpression, ADAR1 activation and a significant reduction in telomere length correlated with accelerated stem cell aging during MPN progression to AML. Increased ADAR1 mediated adenosine to inosine (A-to-I) transcript editing coincided with accelerated telomere shortening in high risk MPN stem cells. Moreover, lentiviral ADAR1 overexpression enhanced pre-LSC engraftment. Treatment with imetelstat reduced MPN stem cell and LSC propagation in stromal co-cultures as well as in humanized mouse models commensurate with reduced hTERT expression levels and telomerase activity and decreased ADAR1 editing activity. Specifically, stromal co-culture assays revealed that combined treatment with dasatinib at 1 nM, and imetelstat at 1 µM or 5 µM significantly inhibited survival and replating of blast crisis (BC) CML progenitors compared with aged bone marrow progenitors (p &amp;lt; 0.001, ANOVA). As a single agent, imetelstat (5 µM) inhibited survival and replating of pre-LSC derived from myelofibrosis compared with normal bone marrow progenitor samples (p &amp;lt; 0.001, ANOVA). In pre-LSC MPN NSG-SGM mouse models established from 4 different MF samples, a significant reduction in proliferation of human CD45+ cells (p &amp;lt; 0.01, t test) was observed in bone marrow and spleen, when compared with vehicle control. Treatment of humanized LSC mouse models, established with 5 different BC CML, with 30 mg/kg of imetelstat, 3 times a week for 4 weeks resulted in a significant reduction in proliferation of malignant progenitors and human CD45+ cells (p &amp;lt; 0.001, ANOVA). As measured by a Flow-FISH assay, abnormal telomere length was reversed by imetelstat treatment compared with mismatch control (p &amp;lt; 0.05, ANOVA). In addition, FACS analysis revealed a significant reduction in activated beta-catenin expression after imetelstat treatment of LSC engrafted mice compared with vehicle control (p &amp;lt; 0.01, ANOVA). Finally, RNA-seq analysis performed on human CD34+ cells from imetelstat treated LSC mouse models revealed a significant reduction in LSC harboring malignant ADAR1-mediated A-to-I editing at doses that spared normal hematopoietic stem cells. CONCLUSIONS Combined WGS and RNA-Seq analyses, lentiviral ADAR1 overexpression, stromal co-culture assays and humanized pre-LSC and LSC mouse model studies reveal that pre-LSC evolution into LSC coincides with both ADAR1 and hTERT activation, which can be prevented with imetelstat. Disclosures Rizo: Geron Corp: Current Employment, Current equity holder in publicly-traded company. Huang:Geron Corp: Current Employment, Current equity holder in publicly-traded company. Jamieson:Forty Seven Inc: Patents &amp; Royalties; Bristol-Myers Squibb: Other.

Choreography of methylation: KDM4C histone demethylation enables ALKBH5 m6A RNA demethylation to sustain AML stem cells.

Choreography of methylation: KDM4C histone demethylation enables ALKBH5 m6A RNA demethylation to sustain AML stem cells.

Resistance Mechanisms to SYK Inhibition in Acute Myeloid Leukemia.

Spleen tyrosine kinase (SYK) is a nonmutated therapeutic target in acute myeloid leukemia (AML). Attempts to exploit SYK therapeutically in AML have shown promising results in combination with chemotherapy, likely reflecting induced mechanisms of resistance to single-agent treatment in vivo. We conducted a genome-scale open reading frame (ORF) resistance screen and identified activation of the RAS-MAPK-ERK pathway as one major mechanism of resistance to SYK inhibitors. This finding was validated in AML cell lines with innate and acquired resistance to SYK inhibitors. Furthermore, patients with AML with select mutations activating these pathways displayed early resistance to SYK inhibition. To circumvent SYK inhibitor therapy resistance in AML, we demonstrate that a MEK and SYK inhibitor combination is synergistic in vitro and in vivo. Our data provide justification for use of ORF screening to identify resistance mechanisms to kinase inhibitor therapy in AML lacking distinct mutations and to direct novel combination-based strategies to abrogate these. SIGNIFICANCE: The integration of functional genomic screening with the study of mechanisms of intrinsic and acquired resistance in model systems and human patients identified resistance to SYK inhibitors through MAPK signaling in AML. The dual targeting of SYK and the MAPK pathway offers a combinatorial strategy to overcome this resistance.This article is highlighted in the In This Issue feature, p. 161.