Therapeutic Strategy For Acute Myeloid Leukemia Research Articles

Acute Myeloid Leukemia: Diagnosis and Evaluation by Flow Cytometry.

With recent technological advances and significant progress in understanding the pathogenesis of acute myeloid leukemia (AML), the updated fifth edition WHO Classification (WHO-HAEM5) and the newly introduced International Consensus Classification (ICC), as well as the European LeukemiaNet (ELN) recommendations in 2022, require the integration of immunophenotypic, cytogenetic, and molecular data, alongside clinical and morphologic findings, for accurate diagnosis, prognostication, and guiding therapeutic strategies in AML. Flow cytometry offers rapid and sensitive immunophenotyping through a multiparametric approach and is a pivotal laboratory tool for the classification of AML, identification of therapeutic targets, and monitoring of measurable residual disease (MRD) post therapy. The association of immunophenotypic features and recurrent genetic abnormalities has been recognized and applied in informing further diagnostic evaluation and immediate therapeutic decision-making. Recently, the evolving role of machine learning models in assisting flow cytometric data analysis for the automated diagnosis and prediction of underlying genetic alterations has been illustrated.

P110CUX1 promotes acute myeloid leukemia progression via regulating pyridoxal phosphatase expression and activating PI3K/AKT/mTOR signaling pathway.

As an evolutionarily conserved transcription factor, Cut-like homeobox 1 (CUX1) plays crucial roles in embryonic and nervous system development, cell differentiation, and DNA damage repair. One of its major isoforms, p110CUX1, exhibits stable DNA binding capabilities and contributes to the regulation of cell cycle progression, proliferation, migration, and invasion. While p110CUX1 has been implicated in the progression of various malignant tumors, its involvement in acute myeloid leukemia (AML) remains uncertain. This study aims to elucidate the role of p110CUX1 in AML. Our findings reveal heightened expression levels of both p110CUX1 and pyridoxal phosphatase (PDXP) in AML cell lines. Overexpression of p110CUX1 promotes AML cell proliferation while inhibiting apoptosis and differentiation, whereas knockdown of PDXP yields contrasting effects. Mechanistically, p110CUX1 appears to facilitate AML development by upregulating PDXP expression and activating the PI3K/AKT/mTOR signaling pathway. Animal experimental corroborate the pro-AML effect of p110CUX1. These results provide experimental evidence supporting the involvement of the p110CUX1-PDXP-PI3K/AKT/mTOR axis in AML progression. Hence, targeting p110CUX1 may hold promise as a therapeutic strategy for AML.

Identification of leukemia stem cell subsets with distinct transcriptional, epigenetic and functional properties

The leukemia stem cell (LSC) compartment is a complex reservoir fueling disease progression in acute myeloid leukemia (AML). The existence of heterogeneity within this compartment is well documented but prior studies have focused on genetic heterogeneity without being able to address functional heterogeneity. Understanding this heterogeneity is critical for the informed design of therapies targeting LSC, but has been hampered by LSC scarcity and the lack of reliable cell surface markers for viable LSC isolation. To overcome these challenges, we turned to the patient-derived OCI-AML22 cell model. This model includes functionally, transcriptionally and epigenetically characterized LSC broadly representative of LSC found in primary AML samples. Focusing on the pool of LSC, we used an integrated approach combining xenograft assays with single-cell analysis to identify two LSC subtypes with distinct transcriptional, epigenetic and functional properties. These LSC subtypes differed in depth of quiescence, differentiation potential, repopulation capacity, sensitivity to chemotherapy and could be isolated based on CD112 expression. A majority of AML patient samples had transcriptional signatures reflective of either LSC subtype, and some even showed coexistence within an individual sample. This work provides a framework for investigating the LSC compartment and designing combinatorial therapeutic strategies in AML.

Synergistic Effects of Glutamine Deprivation and Metformin in Acute Myeloid Leukemia.

The metabolic reprogramming of acute myeloid leukemia (AML) cells is a compensatory adaptation to meet energy requirements for rapid proliferation. This study aimed to examine the synergistic effects of glutamine deprivation and metformin exposure on AML cells. SKM-1 cells (an AML cell line) were subjected to glutamine deprivation and/or treatment with metformin or bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl) ethyl sulfide (BPTES, a glutaminase inhibitor) or cytarabine. Cell viability was detected by Cell Counting Kit-8 (CCK-8) assay, and cell apoptosis and reactive oxygen species (ROS) by flow cytometry. Western blotting was conducted to examine the levels of apoptotic proteins, including cleaved caspase-3 and poly(ADP-ribose) polymerase (PARP). Moreover, the human long noncoding RNA (lncRNA) microarray was used to analyze gene expression after glutamine deprivation, and results were confirmed with quantitative RT-PCR (qRT-PCR). The expression of metallothionein 2A (MT2A) was suppressed using siRNA. Cell growth and apoptosis were further detected by CCK-8 assay and flow cytometry, respectively, in cells with MT2A knockdown. Glutamine deprivation or treatment with BPTES inhibited cell growth and induced apoptosis in SKM-1 cells. The lncRNA microarray result showed that the expression of MT family genes was significantly upregulated after glutamine deprivation. MT2A knockdown increased apoptosis, while proliferation was not affected in SKM-1 cells. In addition, metformin inhibited cell growth and induced apoptosis in SKM-1 cells. Both glutamine deprivation and metformin enhanced the sensitivity of SKM-1 cells to cytarabine. Furthermore, the combination of glutamine deprivation with metformin exhibited synergistic antileukemia effects on SKM-1 cells. Targeting glutamine metabolism in combination with metformin is a promising new therapeutic strategy for AML.

Dual targeting of glutamine and serine metabolism in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy characterized by disrupted blood cell production and function. Recent investigations have highlighted the potential of targeting glutamine metabolism as a promising therapeutic approach for AML. Asparaginases, enzymes that deplete circulating glutamine and asparagine, are approved for the treatment of acute lymphoblastic leukemia, but are also under investigation in AML, with promising results. We previously reported an elevation in plasma serine levels following treatment with Erwinia-derived asparaginase (also called crisantaspase). This led us to hypothesize that AML cells initiate the de novo serine biosynthesis pathway in response to crisantaspase treatment and that inhibiting this pathway in combination with crisantaspase would enhance AML cell death. Here we report that in AML cell lines, treatment with the clinically available crisantaspase, Rylaze, upregulates the serine biosynthesis enzymes phosphoglycerate dehydrogenase (PHGDH) and phosphoserine aminotransferase (PSAT1) through activation of the Amino Acid Response (AAR) pathway, a cellular stress response mechanism that regulates amino acid metabolism and protein synthesis under conditions of nutrient limitation. Inhibition of serine biosynthesis through CRISPR-Cas9-mediated knockout of PHGDH resulted in a ~250-fold reduction in the half-maximal inhibitory concentration (IC50) for Rylaze, indicating heightened sensitivity to crisantaspase therapy. Treatment of AML cells with a combination of Rylaze and a small molecule inhibitor of PHGDH (BI4916) revealed synergistic anti-proliferative effects in both cell lines and primary AML patient samples. Rylaze-BI4916 treatment in AML cell lines led to the inhibition of cap-dependent mRNA translation and protein synthesis, as well as a marked decrease in intracellular glutathione levels, a critical cellular antioxidant. Collectively, our results highlight the clinical potential of targeting serine biosynthesis in combination with crisantaspase as a novel therapeutic strategy for AML.

Epigenetic alterations in AML: Deregulated functions leading to new therapeutic options.

Acute myeloid leukemia (AML) results in disruption of the hematopoietic differentiation process. Crucial progress has been made, and new therapeutic strategies for AML have been developed. Induction chemotherapy, however, remains the main option for the majority of AML patients. Epigenetic dysregulation plays a central role in AML pathogenesis, supporting leukemogenesis and maintenance of leukemic stem cells. Here, we provide an overview of the intricate interplay of altered epigenetic mechanisms, including DNA methylation, histone modifications, and chromatin remodeling, in AML development. We explore the role of epigenetic regulators, such as DNMTs, HMTs, KDMs, and HDACs, in mediating gene expression patterns pushing towards leukemic cell transformation. Additionally, we discuss the impact of cytogenetic lesions on epigenomic remodeling and the potential of targeting epigenetic vulnerabilities as a therapeutic strategy. Understanding the epigenetic landscape of AML offers insights into novel therapeutic avenues, including epigenetic modifiers and particularly their use in combination therapies, to improve treatment outcomes and overcome drug resistance.

An Overview on Lipid Droplets Accumulation as Novel Target for Acute Myeloid Leukemia Therapy.

Metabolic reprogramming is a key alteration in tumorigenesis. In cancer cells, changes in metabolic fluxes are required to cope with large demands on ATP, NADPH, and NADH, as well as carbon skeletons. In particular, dysregulation in lipid metabolism ensures a great energy source for the cells and sustains cell membrane biogenesis and signaling molecules, which are necessary for tumor progression. Increased lipid uptake and synthesis results in intracellular lipid accumulation as lipid droplets (LDs), which in recent years have been considered hallmarks of malignancies. Here, we review current evidence implicating the biogenesis, composition, and functions of lipid droplets in acute myeloid leukemia (AML). This is an aggressive hematological neoplasm originating from the abnormal expansion of myeloid progenitor cells in bone marrow and blood and can be fatal within a few months without treatment. LD accumulation positively correlates with a poor prognosis in AML since it involves the activation of oncogenic signaling pathways and cross-talk between the tumor microenvironment and leukemic cells. Targeting altered LD production could represent a potential therapeutic strategy in AML. From this perspective, we discuss the main inhibitors tested in in vitro AML cell models to block LD formation, which is often associated with leukemia aggressiveness and which may find clinical application in the future.

The Atpase Domain of LONP1 Is Necessary for Mitochondrial Protein Solubility and the Viability of Acute Myeloid Leukemia (AML) Cells

Compared to normal hematopoietic cells, we and others have shown that AML cells have a heightened reliance on oxidative phosphorylation and mitochondrial metabolism for survival and proliferation. To support this unique metabolic phenotype, we previously demonstrated that AML cells have increased import of nuclear-encoded mitochondrial proteins. These newly imported proteins must be properly folded by mitochondrial chaperones, proteases, and heat shock proteins, and failure to properly process and fold these precursors leads to protein aggregation, mitochondrial dysfunction, and cell death. To evaluate the reliance of AML cells on this family of mitochondrial chaperones, proteases, and heat shock proteins, we assessed their dependencies using the gene dependency datasets (eg: depmap.org). From this analysis, we identified the mitochondrial protease, LONP1, as the top hit, and among the top 10% of all essential genes for AML. Localized to the mitochondrial matrix, LONP1 is a nuclear encoded AAA+ serine protease. Proteins are unfolded by its ATPase domain and degraded by its serine-catalyzed proteolytic domain. Compared to normal hematopoietic cells, LONP1 mRNA was overexpressed in AML across three publicly available datasets. Compared to normal hematopoietic cells (n=8) and CD34+ cells (n=3), LONP1 protein was increased in 16/30 primary AML samples by immunoblotting. Using shRNA and CRISPR, LONP1 knockdown and knockout reduced the growth and viability of OCI-AML2, OCI-M2, NB4, and TEX cells. Genetic knockdown or knockout of LONP1 increased levels of insoluble, aggregated mitochondrial proteins as measured by mass spectrometry, proteostat fluorescence and confocal microscopy, and immunoblotting of soluble and insoluble protein fractions. LONP1 knockdown/knockout also reduced mitochondrial respiration, depolarized the mitochondria, and induced AML cell death. We next determined the domain of LONP1 that was necessary for mitochondrial protein solubility, mitochondrial function, and AML survival. We over-expressed wild type, ATPase dead (E591A), or proteolytically dead (S855A) LONP1 cDNA in OCI-AML2 cells and knocked down endogenous LONP1 with shRNA targeting the 3'UTR of the endogenous gene. We then measured mitochondrial protein solubility/aggregation, mitochondrial respiration and cell viability. Wild type LONP1 and the proteolytically dead (S855A) mutant, but not the ATPase mutant (E591A), rescued mitochondrial protein solubility, mitochondrial respiration and cell viability, thus demonstrating that the ATPase domain of LONP1 is necessary for these functions. Bardoxolone methyl (CDDO-Me) is a synthetic triterpenoid that inhibits the ATPase activity of LONP1 by binding to an allosteric site near the ATPase domain of the enzyme. CDDO-Me killed OCI-AML2 and NB4 cells with IC 50 values of 178.5±29.7 and 156.5±39.7 nM, respectively. CDDO-Me (200nM) also killed >50% of cells in 3 out of 4 high-LONP1 expressing primary AML patient samples, but 10 of 10 tested primary AML samples with low levels of LONP1 were insensitive to the drug. Likewise, CDDO-Me induced mitochondrial protein aggregation in OCI-AML2 cells and in a primary AML patient sample with high LONP1 expression, while a primary AML patient sample with undetectable LONP1 expression showed no protein aggregation. In summary, the mitochondrial serine AAA+ protease LONP1 is over-expressed in a subset of AML cells and primary samples. The ATPase domain is necessary for LONP1's function in maintaining mitochondrial protein solubility. Selective inhibition of this domain leads to mitochondrial protein aggregation, impaired mitochondrial respiration and AML cell death. Thus, inhibiting the ATPase domain of LONP1 may be a novel therapeutic strategy for AML.

Enhancer remodeling drives MLL oncogene-dependent transcriptional dysregulation in leukemia stem cells.

Acute myeloid leukemia (AML) with mixed-lineage leukemia (MLL) gene rearrangement (MLLr) comprises a cellular hierarchy in which a subpopulation of cells serves as functional leukemia stem cells (LSCs). They are maintained by a unique gene expression program and chromatin states, which are thought to reflect the actions of enhancers. Here, we delineate the active enhancer landscape and observe pervasive enhancer malfunction in LSCs. Reconstruction of regulatory networks revealed a master set of hematopoietic transcription factors. We show that EP300 is an essential transcriptional coregulator for maintaining LSC oncogenic potential because it controls essential gene expression through modulation of H3K27 acetylation and assessments of transcription factor dependencies. Moreover, the EP300 inhibitor A-485 affects LSC growth by targeting enhancer activity via histone acetyltransferase domain inhibition. Together, these data implicate a perturbed MLLr-specific enhancer accessibility landscape, suggesting the possibility for disruption of the LSC enhancer regulatory axis as a promising therapeutic strategy in AML.

Roles of the hsa_circ_0013880/USP32/Rap1b axis in the proliferation and apoptosis of acute myeloid leukemia cells.

Acute myeloid leukemia (AML) is a myeloid malignancy with generally high mortality. Although recent advances in AML research have revealed that circRNAs play significant roles in AML progression, our understanding of the leukemogenic mechanism of circRNAs remains very limited. In this study, increased expression of hsa_circ_0013880 was observed in bone marrow mononuclear cells (BMNCs) of AML patients. Overexpression of hsa_circ_0013880 promotes AML cell proliferation and migration and reduces cell apoptosis. Mechanistically, hsa_circ_0013880 could elevate the expression of USP32, a deubiquitinating enzyme that is highly expressed in the BMNCs of AML patients. Given the deubiquitination function of USP32, we further hypothesize that USP32 may mediate the malignant behaviors of AML cells by regulating the stability of Ras-related protein (Rap1b). At the molecular level, we find that silencing of USP32 increases ubiquitinated Rap1b. Overexpression of Rap1b restores the effects of USP32 knockdown, which further verifies our hypothesis. In addition, we propose another hypothesis that a potential regulatory network among hsa_circ_0013880, miR-148a-3p/miR-20a-5p and USP32 might exist in the development of AML, according to bioinformatics website predictions and our preliminary experimental verification. Overall, our findings will enrich the understanding of the hsa_circ_0013880/USP32/Rap1b axis in AML development, which may contribute to the development of novel therapeutic strategies for AML.

The m6A reader IGF2BP2 regulates glutamine metabolism and represents a therapeutic target in acute myeloid leukemia.

N6-Methyladenosine (m6A) modification and its modulators play critical roles and show promise as therapeutic targets in human cancers, including acute myeloid leukemia (AML). IGF2BP2 was recently reported as an m6A binding protein that enhances mRNA stability and translation. However, its function in AML remains largely elusive. Here we report the oncogenic role and the therapeutic targeting of IGF2BP2 in AML. High expression of IGF2BP2 is observed in AML and associates with unfavorable prognosis. IGF2BP2 promotes AML development and self-renewal of leukemia stem/initiation cells by regulating expression of critical targets (e.g., MYC, GPT2, and SLC1A5) in the glutamine metabolism pathways in an m6A-dependent manner. Inhibiting IGF2BP2 with our recently identified small-molecule compound (CWI1-2) shows promising anti-leukemia effects invitro and invivo. Collectively, our results reveal a role of IGF2BP2 and m6A modification in amino acid metabolism and highlight the potential of targeting IGF2BP2 as a promising therapeutic strategy in AML.

Dual IKZF2 and CK1α Degrader Targets Acute Myeloid Leukemia Cells

Acute myeloid leukemia (AML) is an aggressive hematologic disease for which several epigenetic regulators have been identified as therapeutic targets. We previously found that a transcription factor and Ikaros family member, IKZF2 (HELIOS) is required for myeloid leukemic stem cell activity (Park et al. Cell Stem Cell 2019). Additionally, IKZF2 enhances self-renewal by increasing HOXA9 gene expression and in parallel blocks myeloid differentiation by reducing C/EBP expression. Thus, our studies suggested that targeting IKZF2 could be a new therapeutic strategy in AML. Immunomodulatory drugs (IMiDs; lenalidomide and others) have been used clinically to specifically target and degrade other Ikaros family members. However, these have been unable to target IKZF2. Utilizing a structure-guided approach, we developed a small molecule with low nanomolar degrader activity against IKZF2 (DEG-35). DEG-35 treatment in different AML cell lines induce apoptosis and differentiation with IC50 values of 5 ±0.4 nM in MOLM-13 cells to 27± 2.5 uM in NB4 cells. To understand if DEG35 has activity against other substrates, we performed global quantitative proteomics and found casein kinase 1 alpha (CK1α), a serine/threonine kinase previously identified to be critical for myeloid leukemogenesis and a known IMiD substrate, to be the most depleted protein (Jaras et al. JEM). Also, we performed a PRISM (Profiling Relative Inhibition Simultaneously in Mixtures) screen assay which utilizes 770 barcoded cell lines from 20 lineages and identified multiple correlations with the p53 pathway. Previous studies found that CK1α suppresses the activity of p53 phosphorylation or binding MDM proteins. To further probe the mechanism behind DEG35 activity, we performed RNA sequencing at 24hrs in MOLM-13 cells. Gene set enrichment analysis (GSEA) using the rank list of differentially expressed genes from the MOLM-13 cells treated with DMSO and DEG-35 revealed enrichment for genes upregulated in CK1α KO keratinocytes and p53 targets. Additionally, we found enrichment for the MLL-AF9 IKZF2 KO LSC gene sets, myeloid differentiation, loss of MEIS1-HOXA9 and MYC targets. Immunoblotting validated the reduction of MYC and HOXA9 protein in DEG-35 treated MOLM-13 cells. To determine the contribution of CK1α and IKZF2 to the cellular phenotypes in MOLM13 cells, we developed CK1α and IKZF2 nondegradable mutants. CK1α, G40N was able to significantly reduce the cell killing activity of DEG-35 by 94% compared to vector cells (**p<0.01). Moreover, p53 deletion was also able to block DEG-35 induced apoptosis by 98% and differentiation by 88% compared to control. Additionally, expression of IKZF2 H141Q rescued DEG-35 mediated differentiation by 55% (***p<0.001), but not apoptosis. Thus, we demonstrate that the degradation of IKZF2 and CK1α by DEG-35 blocks cell growth and induces myeloid differentiation in human AML cell lines through CK1α-p53- and IKZF2-dependent pathways. To further probe the efficacy and therapeutic index of targeting both IKZF2 and CK1α. We found that AML cells were more sensitive to DEG-35 compared to normal bone marrow cells (murine MLL-AF9 Crbn I1391V cells has 10 fold and AML PDX cells have 2-5 fold higher sensitivity to normal mouse or human bone marrow cells).Treatment of DEG-35 at 50 mg/kg in AML PDX-transplanted mice led to significant prolonged survival compared to vehicle treated mice (median survival of DMSO mice: 45 days vs DEG-35 mice: 61 days; ***p< 0.001) and reduced disease burden. To improve pharmacological properties, we developed analog DEG-77, which has 10-fold increased solubility than DEG-35 in DMSO. DEG77 exhibited better in vivo efficacy in the MLL-AF9 Crbn I1391V model (Increase in median survival by DEG-35: 2 days *p<0.05 vs DEG-77: 31 days ****p<0.0001). Importantly, the increase of median survival of 30 days (****p<0.0001) was also observed in the secondarily transplanted DEG-77 mice compared to DMSO mice, suggesting that DEG-35 can reduce LSC activity. In summary, we report the development of nanomolar monofunctional cereblon-dependent degraders of IKZF2 and casein kinase 1 alpha (CK1α). DEG-35 or the analog DEG-77 delays leukemia progression in murine and human AML mice models. Thus, we provide a novel strategy for multi-targeted degradation of IKZF2/CK1α to enhance efficacy against AML that may be expanded to additional targets and indications.

Targeting HIF-Hydroxylases Compromises Disease Initiation and Propagation, and Synergises with Current Therapies to Eliminate Cancer Stem Cells in AML

Acute myeloid leukaemia (AML) is an aggressive disorder of haematopoietic stem and progenitor cells (HSPCs), in which they acquire mutations, resulting in generation of disease fuelling, and ultimately treatment-resistant, leukaemic stem cells (LSCs). Given the hypoxic nature of the bone marrow microenvironment, the role, and pharmacological manipulation of the Hypoxia Inducible Factor (HIF) pathway in normal and malignant haematopoiesis is an area of significant interest. We, and others, previously found that HIF-α, the master regulator of the pathway, is not essential for normal haematopoiesis and its deletion promotes leukaemogenesis. Here we reveal through genetic, pharmacological and omics-based investigations that HIF stabilisation, through inhibition of its regulator PHD2, is a novel potent therapeutic strategy in AML. First, to genetically dissect to role of PHD2 in the initiation of AML, we transformed Phd2cKO HSPCs with AML-associated oncogenes and found that AML cells lacking Phd2 were compromised in vitro, established a weaker leukemic burden and initiated AML with a significantly longer latency in vivo. Moreover, employing our iMLL-AF9;shPhd2 mice, which upon DOX activate MLL-AF9 and a short-hairpin targeting Phd2, we found that Phd2 knockdown in vivo significantly delayed the onset of AML. Next, to investigate acute Phd2 depletion in newly diagnosed disease, we transplanted transformed shPhd2 cells, and upon AML detection induced expression of shPhd2. Notably, Phd2 knockdown in established AML cells compromised disease progression and significantly improved survival of leukaemic mice. Following this, we examined the impact of Phd2 inactivation on HSC function and multilineage haematopoiesis. Phd2cKO mice displayed no adverse phenotypes, and Phd2-deficient HSCs were able to successfully sustain multilineage haematopoiesis upon transplantation. Moreover, global DOX-inducible Phd2 knockdown had little effect on haematopoiesis. Thus, PHD2 inactivation compromises AML development and maintenance, but does not impact normal haematopoiesis, suggesting an ample therapeutic window for its inhibition. Given our genetic evidence that PHD2 is a promising target in AML, we explored the potential of its inhibition. We employed a commercially available clinical trial grade PHD inhibitor, as well as our own novel, selective, and potent PHD inhibitor, both of which work through distinct mechanisms. Significantly, both inhibitors compromised the growth and viability of murine AML cells, as well as multiple established human AML cell-lines and primary patient samples with diverse mutational backgrounds. Moreover, we have also discovered that PHD inhibition markedly synergises with AML treatments currently used in the clinic, thus enhancing their anti-leukaemic effect. Investigating the molecular mechanisms behind our findings, we found HIF-α stabilisation across multiple AML human cell lines following PHD inhibition. Furthermore, we utilised our HifαcKO leukaemic cells which, unlike control cells, were not affected by PHD inhibition, indicating that the anti-leukaemic effect of PHD inhibition is indeed HIF-dependent. Next, we performed RNA-seq and proteomic analyses following genetic and pharmacological PHD inhibition. These experiments revealed increased expression of key myeloid differentiation genes, glycolytic pathway, and a consistent upregulation of HIF target genes, including the pro-apoptotic BNIP3. Finally, pharmacological stimulation of apoptosis combined with PHD inhibition displayed a further and profound anti-leukaemic effect. Taken together, our data strongly indicate that genetic deletion and specific potent pharmacological inhibition of PHD2 can upregulate HIF-dependent pathways, eradicating AML disease in vitro and in vivo, thus unveiling a novel promising therapeutic target.

PIM Inhibition By SEL24/MEN1703 Combines Synergistically with Gilteritinib in FLT3-ITD Preclinical Models of Acute Myeloid Leukemia

BACKGROUND. Acute myeloid leukemia (AML) is an aggressive hematopoietic cancer, derived from an uncontrolled clonal proliferation of malignant myeloid blast cells in the bone marrow, transformed by recurrent genetic alterations (Wu M. et al., J. Hematol Oncol, 2018). SEL24/MEN1703 is a first-in-class, oral, type I dual PIM/FMS-like tyrosine kinase 3 (FLT3) inhibitor investigated in the ongoing Ph2 study in patients with AML (DIAMOND-01, ClinicalTrials.gov identifier: NCT03008187) (Czardybon W. et al., Oncotarget 2018). Gilteritinib is a highly potent and selective oral FLT3 inhibitor, FDA-approved for the treatment of relapsed or refractory AML with FLT3 mutations. Although Gilteritinib showed strong single-agent activity in patients with FLT3+ AML (Levis M. and Perl AE., Blood advances, 2020), the development of Gilteritinib resistance limits durability of the response, indicating that novel combination strategies may be clinically important (Zhang LS. et al., Onco Targets and Therapy, 2022). AIM. The main objective of this study is to assess potential synergistic effects in-vitro and in-vivo of the combination of SEL24/MEN1703 and Gilteritinib in AML cell lines with FLT3-ITD and to elucidate the underlying signaling pathways. METHODS.In-vitro cytotoxicity and apoptosis experiments were carried out in FLT3-ITD AML cell lines (MV4-11, MOLM13, MOLM14), testing SEL24/MEN1703 and Gilteritinib either as single agents or in combination for 72h. The combination index was calculated according to the Chou method (Chou TC., Pharmacol Rev, 2006). In-vitro co-culture experiments were performed with FLT3-ITD cell lines MV4-11 and MOLM14 incubated in the presence of human primary bone marrow stromal cells. In MV4-11 and MOLM13 xenograft models, SEL24/MEN1703 and Gilteritinib were administered per OS every day, for 14 and 22 days, respectively. A signaling pathway analysis (pAKT, pERK, pSTAT5, Mcl-1, c-Myc, Bcl-xL, pS6) was also performed through immuno-capillary electrophoresis on tumor nodules collected from MOLM13 xenograft study. RESULTS. The combination treatment was moderately synergistic/additive in both in-vitro cytotoxicity and apoptosis experiments on MV4-11, MOLM13, and MOLM14, at all concentration levels. However, in co-culture experiments, the presence of primary stromal cells resulted in a significantly increased combination index, in tested AML cell lines. In the in-vivo MV4-11 xenograft model, SEL24/MEN1703 at 25 mg/kg and Gilteritinib at 3 mg/kg induced a significant anti-tumor activity as single agent treatment, showing 57.2% and 84.1% of tumor volume inhibition (TVI) respectively, whereas the combination treatment SEL24/MEN1703 plus Gilteritinib induced 99.2% of TVI at day 33 (Figure 1). At day 40, when the Gilteritinib schedule was concluded, SEL24/MEN1703 and Gilteritinib as single agents showed 53.1% and 87.6% of TVI respectively, whereas the combo group induced a complete tumor regression (100% of TVI). Moreover, only the combination of SEL24/MEN1703 and Gilteritinib resulted in a delayed tumor regrowth 23.5 days after discontinuation of the treatment. In the MOLM13 model, at day 29, treatment with SEL24/MEN1703 at 25 mg/kg and Gilteritinib at 3 mg/kg as a monotherapy induced an antitumor activity with 38.8% and 60.1% of TVI respectively, whereas with the combination treatment resulted in a higher TVI (78.3%), however, no complete tumor regression was observed in this model. In the MOLM13 xenograft model, analysis of downstream signaling pathways of FLT3 and PIM kinases showed that the pS6 effector protein is mainly downregulated in the combination treatment group. CONCLUSIONS. Combination therapy with SEL24/MEN1703 and Gilteritinib is moderately synergistic in-vitro, however, results in robust tumor regressions, including complete responses in in-vivo FLT3-ITD models, at tolerable doses. The higher efficacy of this combination in-vivo and compared to monotherapy could be related to a very potent concomitant inhibition of FLT3 and PIM kinases affecting upregulated FLT3 signaling pathway in-vivo as demonstrated by the reduction of phosphorylation of downstream effector proteins, such as pS6. Overall, these experiments have demonstrated the antitumor potential of concomitant inhibition of FLT3 and PIM kinases which could drive a novel therapeutic strategy in AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract 3023: Fatty acid synthase (FASN) inhibitors synergize with carfilzomib (CFZ) in acute myeloid leukemia (AML) and multiple myeloma (MM)

Abstract Background: Fatty acid synthase (FASN) upregulation during conditions of oxidative stress contributes to tumor proliferation and survival, which appears to be a mechanism of proteasome inhibitor resistance. Here we demonstrate that acute myeloid leukemia (AML) cell lines resistant to carfilzomib (CFZ), a second-generation proteasome inhibitor, have higher basal FASN expression and targeting FASN with small molecule inhibitors enhances the cytotoxic effect of CFZ in both AML and multiple myeloma (MM) cell lines. Methods: In a proliferation assay, human AML and MM cell lines were treated with a single dose of CFZ for 7 days. Inhibition of proliferation was defined using an IC50 cutoff for CFZ of 10nM for AML and 5nM for MM as the threshold for sensitivity. Sensitive and resistant cell lines were subjected to apoptosis and cell cycle analyses by flow cytometry after being exposed to CFZ for 72 hours. Proteomic analysis was performed at baseline using reverse phase protein assay (RPPA). For CFZ and FASN inhibitor combination assays, AML and MM cell lines with varying sensitivities to CFZ were exposed to CFZ and FASN inhibitors, orlistat or TVB-3166, simultaneously, and the apoptosis rate were analyzed by flow cytometry. For western blots, selected AML and MM cell lines were incubated with compounds for 24 hours, and the lysates were probed for selected targets. Results: Single-agent CFZ induced apoptosis in sensitive AML and MM cell lines, while apoptotic rates remained low in resistant cell lines. Cell cycle analysis showed increased sub-G1 population in sensitive cell lines compared to resistant cell lines. RPPA revealed that FASN, a key enzyme involved in lipogenesis, correlated with CFZ sensitivity, and CFZ resistant lines trended towards higher basal FASN levels. When CFZ was combined with FASN inhibitors, orlistat or TVB-3166, significant synergy was observed in the apoptosis assays in the AML and MM cell lines. Western blot analyses showed FASN inhibitors enhanced the anti-proliferation and pro-apoptotic effects of CFZ. Conclusion: CFZ demonstrated single agent activity in nanomolar ranges in human AML and MM cell lines. When combined with agents targeting lipid-metabolism, CFZ showed synergistic effect in apoptosis, suggesting this combination could potentially be a new therapeutic strategy for AML and MM. Citation Format: Maoyu Peng, Sanaz Noelle Ghafouri, Martina SJ McDermott, Dennis J. Slamon, Sarah M. Larson. Fatty acid synthase (FASN) inhibitors synergize with carfilzomib (CFZ) in acute myeloid leukemia (AML) and multiple myeloma (MM) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3023.

Ex vivo isolation, expansion and bioengineering of CCR7+CD95-/or CD62L+CD45RA+ tumor infiltrating lymphocytes from acute myeloid leukemia patients' bone marrow.

T cell based immunotherapies can be applicable to acute myeloid leukemia (AML). Therefore, the selection of optimal T cells, cell manufacturing, and therapeutic T cell engineering are essential for the development of effective adoptive T cell therapies for AML. Autologous tumor-infiltrating lymphocytes (TILs) have been in clinical trials to treat solid malignancies. Herein, we assessed whether TILs can be isolated from the bone marrow (BM) of AML patients, expanded ex vivo and utilized as a novel therapeutic strategy for AML. To this end, firstly we analyzed the immunophenotypes of a series of primary BM samples from AML patients (N = 10) by flow cytometry. We observed a variable amount of CD3+ TILs (range ∼2.3–∼32.6% of mononuclear cells) among BM samples. We then developed a novel protocol that produced a three-log ex vivo expansion of TILs isolated from AML patient BM (N = 10) and peripheral blood (PB) (N = 10), including from patients with a low number of CD3+ T cells, within 3, 4 weeks. Further, we identified previously described naïve T cells (CCR7+CD95-/or CD62L+CD45RA+) in AML BM and PB samples, which seemed to be required for a successful TILs ex vivo expansion. Finally, we showed that the expanded TILs could: (1) cause cytotoxicity to autologous AML blasts ex vivo (90.6% in control without T cell treatment vs. 1.89% in experimental groups with PB derived T cells and 1.77% in experimental groups with BM derived TILs, p < 0.01), (2) be genetically engineered to express CYP27B1 gene, and (3) infiltrate the BM and reside in close proximity to pre-injected autologous AML blasts of engrafted immunodeficiency mice. Altogether, these results provide a rationale for further studies of the therapeutic use of TILs in AML.

Validation of NMT1 and NMT2 As Novel Drug Targets in Adult Acute Myeloid Leukemia: Rationale for N-Myristoyltransferase Inhibition with Pclx-001 for Clinical Trials

Background: N-terminal myristoylation is the transfer of the saturated fourteen carbon fatty acid myristate to an N-terminal glycine residue. This co- or post-translational protein modification promotes protein-protein and protein-lipid interactions and is essential for proper membrane localization and/or activity of up to 600 human intracellular proteins. N-myristoyltransferases (NMTs) are the enzymes responsible and two isoforms are found in humans. NMT1 (ubiquitous and essential for cell survival) and NMT2 (more variably expressed) differ in activity level and substrate specificities. NMT expression levels vary in some cancers, and with myristoylation being essential for activity of certain oncogenes including Src Family Kinases (SFKs). NMTs have therefore been proposed as an anti-cancer target. Dysregulation and oncogenic activity of SFKs occurs frequently in acute myeloid leukemia (AML), suggesting NMT inhibition could provide therapeutic benefit. PCLX-001 is a low nanomolar small molecule pan-NMT inhibitor with high oral bioavailability in clinical trials as once daily oral therapy for lymphoma and solid tumors.Methods and Results: Data from the TCGA Transcriptome database showed high NMT1 and low NMT2 were associated with reduced overall and event-free survival in adult AML, and high NMT1 - but not NMT2 - expression is associated with proliferative gene sets in AML cell lines. AML cell lines treated with PCLX-001 showed a significant reduction in total protein myristoylation, as well as reduced levels of SFK proteins and SFK phosphorylation. PCLX-001 induced apoptosis in AML cell lines and patient blasts at concentrations which spared a large proportion of peripheral blood lymphocytes and monocytes from healthy individuals. AML cell lines showed significant increase in BIP protein and ER stress in response to PCLX-001, along with caspase 3 cleavage. In an AML cell line derived xenograft (CDX) and two AML patient derived xenograft (PDX) series (n=1 DX for MV-4-11 and n=2 PDX), PCLX-001 monotherapy had dose-dependent anticancer activity and resulted in complete remissions in subcutaneous AML cell deposits. In tail-vein injection PDX models, PCLX-001 treatment resulted in up to 95% reduction of human CD45+ cells in peripheral blood and bone marrow.Conclusions: These findings validate NMT inhibition as a novel therapeutic strategy for AML. PCLX-001 preferentially targeted AML cells that rely on oncogenic activity of myristoylated proteins, inducing apoptosis and reducing leukemic burden. PCLX-001 warrants evaluation in clinical trials for adult AML. DisclosuresGamma: Pacylex Pharmaceuticals: Current holder of individual stocks in a privately-held company. Yap: Pacylex Pharmaceuticals: Current holder of individual stocks in a privately-held company, Patents & Royalties. Beauchamp: Pacylex Pharmaceuticals: Current Employment, Current holder of individual stocks in a privately-held company, Patents & Royalties. Mackey: Pacylex Pharmaceuticals, Inc.: Current holder of individual stocks in a privately-held company. Pemmaraju: Dan's House of Hope: Membership on an entity's Board of Directors or advisory committees; Abbvie Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other, Research Funding; Aptitude Health: Consultancy; Sager Strong Foundation: Other; Celgene Corporation: Consultancy; LFB Biotechnologies: Consultancy; Plexxicon: Other, Research Funding; MustangBio: Consultancy, Other; Roche Diagnostics: Consultancy; Daiichi Sankyo, Inc.: Other, Research Funding; DAVA Oncology: Consultancy; Cellectis S.A. ADR: Other, Research Funding; Springer Science + Business Media: Other; Stemline Therapeutics, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other, Research Funding; HemOnc Times/Oncology Times: Membership on an entity's Board of Directors or advisory committees; ASCO Leukemia Advisory Panel: Membership on an entity's Board of Directors or advisory committees; Samus: Other, Research Funding; ASH Communications Committee: Membership on an entity's Board of Directors or advisory committees; CareDx, Inc.: Consultancy; Novartis Pharmaceuticals: Consultancy, Other: Research Support, Research Funding; Incyte: Consultancy; Affymetrix: Consultancy, Research Funding; Protagonist Therapeutics, Inc.: Consultancy; Clearview Healthcare Partners: Consultancy; Blueprint Medicines: Consultancy; Bristol-Myers Squibb Co.: Consultancy; ImmunoGen, Inc: Consultancy; Pacylex Pharmaceuticals: Consultancy. Borthakur: Astex: Research Funding; Ryvu: Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Protagonist: Consultancy; ArgenX: Membership on an entity's Board of Directors or advisory committees; University of Texas MD Anderson Cancer Center: Current Employment; Takeda: Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy. Brandwein: AbbVie: Honoraria; Jazz: Honoraria; Taiho: Honoraria; Astellas: Honoraria; Bristol Myers Squibb: Honoraria; Roche: Honoraria; Pfizer: Honoraria; Amgen: Honoraria. Berthiaume: Pacylex Pharmaceuticals, Inc.: Current holder of individual stocks in a privately-held company.

Evaluation of Poly(ADP-ribose) Polymerase Inhibitor, Pamiparib (BGB-290) in Treating Acute Myeloid Leukemia and the Characterization of Its Nanocarrier.

Despite the continuous improvement of leukemia treatment in the clinic, the overall 5-year disease-free survival of acute myeloid leukemia (AML) is only approximately 30%-60% due to relapse and the refractoriness of AML after traditional chemotherapy. Inhibition of poly(ADP-ribose) polymerase (PARP), a member of the DNA damage repair complex, has a strong antitumor effect in solid tumors. However, the role of PARP in AML remains unclear. We found that high levels of PARP1 and PARP2 were positively related to chemotherapy resistance and poor prognosis in patients with AML. Doxorubicin (DOX)-resistant AML cells highly expressed PAPR1 and PARP2. Knockdown of PARP1 and PARP2, or pharmaceutical inhibition of PARP by the PARP inhibitor (PARPi) BGB-290, significantly enhanced the cytotoxicity of DOX in AML cells due to increased DNA damage. PLGA-loading BGB-290 was properly self-assembled into stable BGB-290@PLGA nanoparticles (NPs), which is uniform particle size and good stability. BGB-290@PLGA is easily uptake by AML cell lines and stays for a long time. Combined with DOX, BGB-290@PLGA can significantly improve the chemosensitivity of AML cell lines. Furthermore, BGB-290 and DOX combination treatment dramatically repressed the onset of leukemia and prolonged the survival of THP-1 xenografted mice. Overall, this study demonstrated that PARPi with traditional chemotherapy could be an efficient therapeutic strategy for AML.

Patient Heterogeneity in Acute Myeloid Leukemia: Leukemic Cell Communication by Release of Soluble Mediators and Its Effects on Mesenchymal Stem Cells.

Acute myeloid leukemia (AML) is an aggressive bone marrow malignancy, and non-leukemic stromal cells (including mesenchymal stem cells, MSCs) are involved in leukemogenesis and show AML-supporting effects. We investigated how constitutive extracellular mediator release by primary human AML cells alters proteomic profiles of normal bone marrow MSCs. An average of 6814 proteins (range 6493−6918 proteins) were quantified for 41 MSC cultures supplemented with AML-cell conditioned medium, whereas an average of 6715 proteins (range 6703−6722) were quantified for untreated control MSCs. The AML effect on global MSC proteomic profiles varied between patients. Hierarchical clustering analysis identified 10 patients (5/10 secondary AML) showing more extensive AML-effects on the MSC proteome, whereas the other 31 patients clustered together with the untreated control MSCs and showed less extensive AML-induced effects. These two patient subsets differed especially with regard to MSC levels of extracellular matrix and mitochondrial/metabolic regulatory proteins. Less than 10% of MSC proteins were significantly altered by the exposure to AML-conditioned media; 301 proteins could only be quantified after exposure to conditioned medium and 201 additional proteins were significantly altered compared with the levels in control samples (153 increased, 48 decreased). The AML-modulated MSC proteins formed several interacting networks mainly reflecting intracellular organellar structure/trafficking but also extracellular matrix/cytokine signaling, and a single small network reflecting altered DNA replication. Our results suggest that targeting of intracellular trafficking and/or intercellular communication is a possible therapeutic strategy in AML.

MiR-143 Targets ERK5 in Granulopoiesis and Predicts Outcome of Patients with Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a very heterogeneous disease of the hematopoietic system which is characterized by a fast progress with poor survival options. Despite intensive research standard treatment strategies rely since decades on the same chemotherapeutics, and the overall survival options still remain poor.In recent years, there is an emerging role of microRNAs (miRNAs) in the regulation of cancer development and resistance to cancer therapies. Mechanistically, miRNAs regulate gene expression through modulation of multiple target mRNAs by mRNA degradation or block of translation. It is shown that miRNAs can act as tumor suppressors and oncogenes dependent on the type of cancer. The miRNA-143/145 cluster is shown to be down-regulated in most cancers and is able to modulate tumorigenesis by targeting tumor associated genes. Several miR-143/145 targets are well described oncogenes, such as ERK5, DNMT3A or Bcl2. Interestingly, recent studies showed that the miR-143/145 cluster can regulate multidrug resistance proteins (MDR) in various cancer types and that enforced expression of miR-143/145 leads to increased sensitivity to various drugs. Besides its well-studied function mainly in solid tumors the role of the miR-143/145 cluster or miR-143 alone especially in the hematopoietic system remains largely unknown.In this study, we report a previously unrecognized function of miR-143 in granulopoiesis. Based on our observation that hematopoietic cells undergoing granulocytic differentiation exhibit increased miR-143 expression, we show that the absence of miR-143 in mice results in a reduced number of mature granulocytes but not monocytes in blood and bone marrow. Additionally, we show that overexpression or ablation of miR-143 resulted in accelerated granulocytic differentiation or block of differentiation in several in vitro systems, including leukemic cell lines and G-CSF treated CD34+ HSPCs, respectively.To identify the mRNA targets affected by miR-143 in hematopoietic cells we performed Argonaut2-RNA-Immunoprecipitation coupled with next generation sequencing. Here we could demonstrate a strong enrichment of members of the MAPK- and mTOR-pathways in cells overexpressing miR-143. Besides several potential targets of miR-143 we found ERK5 as particularly interesting since ERK5 was found to be a central mediator of cell survival, proliferation, motility, differentiation and apoptotic regulation of normal cells. To test whether miR-143 overexpression would also affect ERK5 protein abundance we performed western blot analysis of ERK5 and activated ERK5 in hematopoietic cells infected with miR-143 O/E constructs. Here we could show that miR-143 leads to reduced ERK5 protein levels in these cells. Since deregulation of both factors miR-143 as well as ERK5 have been implicated in the response to chemotherapeutic agents we analyzed the impact of miR-143 expression on treatment response. Here we show that AML patients receiving 5-azacytidine (Vidaza) and respond to the therapy had significant higher miR-143 levels than patients which did not respond. To test whether miR-143 expression would affect the response to chemotherapeutics, we overexpressed miR-143 in leukemic cells followed by Vidaza and AC220 treatment. Our results show a significant increase of apoptosis in cells overexpressing miR-143. Finally, we observed an association of high miR-143 expression levels with a higher probability of survival in two different cohorts of patients with acute myeloid leukemia. Taken together, we identified miR-143 as a relevant factor in granulocytic differentiation, which expression may be useful as prognostic factor and as new target for therapeutic strategies in AML. DisclosuresSchwind:Novartis: Consultancy.