Strategy For Acute Myeloid Leukemia Research Articles

Deep PIM kinase substrate profiling reveals new rational cotherapeutic strategies for acute myeloid leukemia

AbstractProvirus integration site for Moloney murine leukemia virus (PIM) family serine/threonine kinases perform protumorigenic functions in hematologic malignancies and solid tumors by phosphorylating substrates involved in tumor metabolism, cell survival, metastasis, inflammation, and immune cell invasion. However, a comprehensive understanding of PIM kinase functions is currently lacking. Multiple small-molecule PIM kinase inhibitors are currently being evaluated as cotherapeutics in patients with cancer. To further illuminate PIM kinase functions in cancer, we deeply profiled PIM1 substrates using the reverse in-gel kinase assay to identify downstream cellular processes targetable with small molecules. Pathway analyses of putative PIM substrates nominated RNA splicing and ribosomal RNA (rRNA) processing as PIM-regulated cellular processes. PIM inhibition elicited reproducible splicing changes in PIM-inhibitor–responsive acute myeloid leukemia (AML) cell lines. PIM inhibitors synergized with splicing modulators targeting splicing factor 3b subunit 1 (SF3B1) and serine-arginine protein kinase 1 (SRPK1) to kill AML cells. PIM inhibition also altered rRNA processing, and PIM inhibitors synergized with an RNA polymerase I inhibitor to kill AML cells and block AML tumor growth. These data demonstrate that deep kinase substrate knowledge can illuminate unappreciated kinase functions, nominating synergistic cotherapeutic strategies. This approach may expand the cotherapeutic armamentarium to overcome kinase inhibitor–resistant disease that limits durable responses in malignant disease.

Anlotinib synergizes with venetoclax to induce mitotic catastrophe in acute myeloid leukemia

Venetoclax is a BCL2-targeted drug employed in treating various cancers, particularly hematologic malignancies. Venetoclax combination therapies are increasingly recognized as promising treatment strategies for acute myeloid leukemia (AML). In this study, we conducted an unbiased drug screen and identified anlotinib, a promising multi-targeted receptor tyrosine kinase inhibitor with oral activity currently utilized in the treatment of solid tumor, as a potent enhancer of venetoclax's anticancer activity in AML. Our investigation encompassed AML cell lines, primary cells, and mouse models, demonstrating effective low-dose combination therapy of anlotinib and venetoclax with minimal cytopenia or organ damage. Proteomic analysis revealed abnormal mitotic signals induced by this combination in AML cells. Mechanistically, anlotinib synergized with venetoclax by suppressing ARPP19 protein, leading to sustained activation of PP2A-B55δ. This inhibited AML cells from entering the mitotic phase, culminating in mitotic catastrophe and apoptosis. Additionally, we identified a specific synthetic lethal vulnerability in AML involving an ARPP19 mutation at S62 phosphorylation. These findings underscore the therapeutic potential of anlotinib and venetoclax combination therapy in AML, warranting further clinical investigation.

Reduced dose azacitidine plus venetoclax as maintenance therapy in acute myeloid leukaemia following intensive or low-intensity induction: a single-centre, single-arm, phase 2 trial

Reduced dose azacitidine plus venetoclax as maintenance therapy in acute myeloid leukaemia following intensive or low-intensity induction: a single-centre, single-arm, phase 2 trial

Mathematical modelling of clonal reduction therapeutic strategies in acute myeloid leukemia

Over the years, the overall survival of older patients diagnosed with acute myeloid leukemia (AML) has not significantly increased. Although standard cytotoxic therapies that rapidly eliminate dividing myeloblasts are used to induce remission, relapse can occur due to surviving therapy-resistant leukemic stem cells (LSCs). Hence, anti-LSC strategies have become a key target to cure AML. We have recently shown that previously approved cardiac glycosides and glucocorticoids target LSC-enriched CD34+ cells in the primary human AML 8227 model with more efficacy than normal hematopoietic stem cells (HSCs). To translate these in vitro findings into humans, we developed a mathematical model of stem cell dynamics that describes the stochastic evolution of LSCs in AML post-standard-of-care. To this, we integrated population pharmacokinetic-pharmacodynamic (PKPD) models to investigate the clonal reduction potential of several promising candidate drugs in comparison to cytarabine, which is commonly used in high doses for consolidation therapy in AML patients. Our results suggest that cardiac glycosides (proscillaridin A, digoxin and ouabain) and glucocorticoids (budesonide and mometasone) reduce the expansion of LSCs through a decrease in their viability. While our model predicts that effective doses of cardiac glycosides are potentially too toxic to use in patients, simulations show the possibility of mometasone to prevent relapse through the glucocorticoid’s ability to drastically reduce LSC population size. This work therefore highlights the prospect of these treatments for anti-LSC strategies and underlines the use of quantitative approaches to preclinical drug translation in AML.

Dendritic Cells as a Therapeutic Strategy in Acute Myeloid Leukemia: Vaccines.

Dendritic cells (DCs) serve as professional antigen-presenting cells (APC) bridging innate and adaptive immunity, playing an essential role in triggering specific cellular and humoral responses against tumor and infectious antigens. Consequently, various DC-based antitumor therapeutic strategies have been developed, particularly vaccines, and have been intensively investigated specifically in the context of acute myeloid leukemia (AML). This hematological malignancy mainly affects the elderly population (those aged over 65), which usually presents a high rate of therapeutic failure and an unfavorable prognosis. In this review, we examine the current state of development and progress of vaccines in AML. The findings evidence the possible administration of DC-based vaccines as an adjuvant treatment in AML following initial therapy. Furthermore, the therapy demonstrates promising outcomes in preventing or delaying tumor relapse and exhibits synergistic effects when combined with other treatments during relapses or disease progression. On the other hand, the remarkable success observed with RNA vaccines for COVID-19, delivered in lipid nanoparticles, has revealed the efficacy and effectiveness of these types of vectors, prompting further exploration and their potential application in AML, as well as other neoplasms, loading them with tumor RNA.

Abstract B174: The non-covalent HCK/BTK inhibitor TTX-114 displays potent in vitro and in vivo anti-tumor activity in leukemia and lymphoma

Abstract Background: The SRC family kinase Hematopoietic Cell Kinase (HCK) is upregulated in cycle–quiescent and chemotherapy-resistant leukemic stem cells (LSCs), and inhibition of HCK reduces the survival and self-renewal capacity of LSCs. As such, targeting HCK has emerged as a potential therapeutic strategy in acute myeloid leukemia (AML). In lymphoma, HCK phosphorylates and activates Bruton Tyrosine Kinase (BTK) downstream of Toll-like receptor (TLR) or the IL6 receptor. In conjunction with BTK, HCK engages essential pro-survival signaling through the NF-kB, mTOR, AKT and STAT pathways. Meanwhile, in lymphoma, HCK can take over pro-survival signaling to confer resistance to BTK inhibition. A dual HCK/BTK inhibitor thus provides a promising therapeutic approach to target leukemias and lymphomas. Methods: TTX-114 was identified through a structure-based medicinal chemistry campaign and characterized in cells using Western Blot and Cell Titer Glow for signaling and viability read-outs, respectively. For in vivo efficacy analysis, NSG newborn mice were irradiated prior to bone-marrow reconstitution with patient-derived primary AML LCSs. Mice were treated orally with TTX-114 after AML was established. Results: Here, we describe TTX-114, a non-covalent HCK/BTK inhibitor with anti-tumor activity in leukemia and lymphoma. In biochemical kinase profiling TTX-114 displayed sub-nM activity against BTK with a KD = 0.58 nM and the SRC-kinase cluster with a KD = 0.23nM for HCK. By inhibiting HCK and BTK activity in cells, TTX-114 prevented autophosphorylation of HCK at Y411 and BTK at Y223 in a dose-dependent manner in human peripheral blood mononuclear cells (PBMCs), enabling a starting point for clinical demonstration of target engagement. TTX-114 had anti-tumor activity across a larger panel of leukemia and lymphoma cell lines, with diffuse large B cell lymphoma (DLBCL) cell lines being among the most sensitive (IC50, ≤4 nM). In marginal zone lymphoma (MZL), the activity was slightly higher in the presence of MYD88 L265P mutation. In vivo, TTX-114 treatment significantly reduced the tumor burden in a dose and time-dependent manner in AML-derived PDX mice. A systematic pharmacokinetic/pharmacodynamic (PK/PD) study identified distinct PK driver for efficacy and tolerability, respectively enabling a well-tolerated dosing strategy for TTX-114 which induced complete response in AML-xenograft bearing mice with no impact on body weight. Conclusion: TTX-114 is an orally bioavailable, non-covalent HCK/BTK inhibitor which prevents HCK over-activation in cancer cells and provides a new targeted therapy for leukemias and lymphomas. Citation Format: Ulrike Rauh, Michael Serrano-Wu, Virendar Kaushik, Sabine Pilari, Ikuko Ogahara, Akiko Kaneko, Alberto J Arribas, Eleonora Cannas, Franceso Bertoni, Nathalie R Javidi-Sharifi, Matthew S Davids, Takehiro Fukami, Yoriko Saito, Fumihiko Ishikawa, Anthony Letai, Brian Hubbard. The non-covalent HCK/BTK inhibitor TTX-114 displays potent in vitro and in vivo anti-tumor activity in leukemia and lymphoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B174.

Metabolism-Related Features Identify the Combination Metformin Plus NAMPT Inhibitors As a Selective Treatment Strategy in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a heterogeneous disease, which remains difficult to treat due to its heterogeneous nature. Recently, metabolic adaptation has been recognized as a trademark of leukemogenesis. Notably, altered function of mitochondria (mt) in leukemic stem cells (LSCs) has been linked to chemoresistance in some patients, causing relapse of disease. Thus, targeting the rewired energy metabolism of leukemic cells has gained a lot of attention in recent years. Here, using a large cohort of adult de novo AML samples (n=502 patients, all intensively treated with 3+7 scheme), we identified a subset of patients with increased mitochondrial DNA content (mtDNAcn, n=163) compared to age- and sex-matched healthy controls (n=308). mtDNAcn was significantly higher in patients harboring FLT3-ITD (31%) and IDH1 mutations (50%). Patients with high mtDNAcn were associated with worse clinical outcomes regardless of European Leukemia Net (ELN) risk stratification, sex, and age. In contrast, high mtDNAcn was not a predictive survival marker in a cohort of adult AML patients treated with venetoclax (VEN) + hypomethylating agents (HMA), indicating that these metabolic features are specifically linked to the resistance to intensive chemotherapy. Using label-free quantitative proteome analysis (11272 proteins) combined with metabolomic screening (173 metabolites) on sorted CD34 +/CD117 + AML blasts (n=26), we found that AMLs with high mtDNAcn were enriched for signatures like “GMP-like” and “mt complex I activity”, being metabolically enriched for long-chain fatty acids, suggesting that these cells rely more on oxidative phosphorylation (OXPHOS) metabolism. Functionally, AML blasts with high mtDNAcn displayed increased levels of BCL2, mt mass, and mt membrane potential, which was associated with increased oxygen consumption rate (OCR) measured by extracellular flux analysis. Considering the impact of mtDNAcn on AML survival in patients treated with intensive chemotherapy, which was not observed in the context of VEN+HMA, and considering the increased reliance of LSC on mtOXPHOS, we studied the impact of mtDNAcn on chemotherapy response. Ex vivo analysis of primary AML samples with high mtDNAcn indicated increased resistance to AraC-induced apoptosis, which was reverted upon knock-down of the DNA polymerase gamma gene (POLG) encoding the catalytic subunit of the mtDNA polymerase. Due to the link between mtDNAcn and mt complex I activity, we investigated if the treatment with metformin, known to inhibit complex I, would sensitize AML patients with high mtDNAcn to the standard of care AML drugs, such as Cytarabine (AraC), FLT3-inhibitors and VEN. Treatment with low dose of metformin (1 mM) effectively reduced the mtDNAcn in AML cells (n=40), resulting in decreased OCR, while glycolysis was increased, suggesting metabolic rewiring. Furthermore, the combination of metformin with AraC, VEN and FLT3-inhibitors synergistically increased drug-induced apoptosis in AML cells with high mtDNAcn and helped to overcome VEN resistance in AML cells. In vivo, combination of metformin with VEN prolonged the survival of mice transplanted with K562 cells (VEN resistant), resulting in decreased leukocyte counts and blood chimerism. Since treatment with metformin resulted in metabolic rewiring, with increased glycolysis and NAD + consumption, we wondered if the combination of metformin with glycolytic (2-DG/DAP) and NAMPT (KPT-9274) inhibitors would impede the metabolic rewiring, enhancing cytotoxicity. Among the different treatment combinations, we find that the combination of metformin and KPT-9274 gave the best therapeutic window by significantly increasing drug-induced apoptosis, with more pronounced effects in patients with high mtDNAcn, while sparing healthy CD34 + cells. Concomitantly, OXPHOS and glycolysis were diminished upon KPT-9274+metformin, suggesting that the combination was able to bypass the metabolic rewiring of the AML cells. In conclusion, we uncover a subgroup of patients with high mtDNAcn linked to increased mtOXPHOS that is resistant to chemotherapy-induced apoptosis and is characterized by poor clinical outcomes, which can be overcome by the inhibition of the mt complex I. Moreover, the quantification of mtDNAcn can be easily incorporated into clinical practice as a simple and cost-effective metabolic readout of AML patients to identify metabolic vulnerabilities.

Single-Cell Analyses Unveil a Potential Mechanism Promoting Graft-Versus-Leukemia Response in Haploidentical Transplantation Using Peripheral Stem Cells Plus Cord Blood Grafts through Early Immune Reconstruction of CD8+ Infg+ T Cells

Abstract Background Haploidentical donor stem cell transplantation (haplo-SCT) by using G-CSF-primed peripheral stem cells combined with bone marrow grafts (PBSC+BM) represents a widely employed strategy for acute myeloid leukemia (AML). While leukemic relapse is one of the main reasons for the failure of SCT. Surprisingly, our clinical data found that haploidentical peripheral stem cells combined with cord blood (haplo-PBSC+cord) transplantation had significantly lower incidence of relapse than haplo-PBSC+BM transplantation. Nonetheless, the underlying mechanism needs to be explored. Method From 2021 to 2022, bone marrow samples at 1 and 3 months post-transplantation from 4 AML patients who underwent haplo-PBSC+cord transplantation and 3 AML patients who received haplo-PBSC+BM transplantation were obtained. We used single-cell RNA-sequence (scRNA-seq) technology for analysis of hematopoietic reconstitution disparity between the PBSC+cord and PBSC+BM groups. Results Significant disparities manifest in the initial stages of hematopoietic reconstitution between the PBSC+cord and the PBSC+BM groups. Notably, at 1 month and 3 months post-transplantation, the PBSC+cord group demonstrated conspicuous reconstitution of lymphoid and erythroid cells, with CD8+ T cells exhibiting the most pronounced effect. Differential gene analysis revealed that T cells in the PBSC+cord group display elevated expression of graft-versus-leukemia (GVL)-related cytokines, including IFN-γ, CCL3, and IL1B, when compared to the PBSC+BM group. Furthermore, noteworthy differences in T cell subsets were observed between the two groups. Within 1 month and 3 months post-transplantation, the PBSC+cord group experienced up-regulation of CD8+ effector memory T cells (TEM), characterized by heightened expression of the IFNG gene. Gene Set Variation Analysis (GSVA) unveiled significant enrichment of CD8+ IFNG+ TEM in T cell activation, cytotoxicity, cytokine secretion, and other pathways, suggesting its potential GVL effect. On the contrary, the PBSC+BM group demonstrated a higher proportion of CD4+ T cells, γδT cells, and MAIT cells at 1 month and 3 months post-transplantation. Among CD8+ T cells, the PBSC+BM group showcased a more robust reconstruction of CD8+ TIGIT+ T cell subsets, which highly express exhaustion-related genes like TIGIT, LAG3, PDCD1 (PD-1), HAVCR2 (TIM-3), and exhibited increased apoptosis-related pathway activity. Conclusion Our investigation identified a potential mechanism promoting GVL response in the immune reconstitution of haplo-PBSC+cord transplantation.

NGS Profile and the Mathematical Prediction Model for Venetoclax Combination Therapy in HM-Screen-Japan 02 Study

[Introduction] Azacitidine and venetoclax combination therapy (Aza/Ven) is a novel strategy for acute myeloid leukemia (AML). Although this regimen is widely used as a first line treatment not only for unfit/elderly patients but also for fit/young patients in the real-world situation, appropriate patient selection is an important issue. HM-SCREEN-Japan 02 (UMIN-CTR UMIN000046371) is a Japanese multicenter prospective observational study to evaluate the clinical utility of targeted sequencing. In this study, we performed the sub-analysis of newly diagnosed AML patients who received Aza/Ven as a 1 st line treatment to elucidate the genetic background of AML. Furthermore, we proposed a novel mathematical model to predict the response of Aza/Ven. [Methods] AmoyDx® Myeloid Blood Cancer Panel for acute myeloid leukemia was used for the targeted sequencing. We performed linear discriminant analysis to predict complete response patients by Aza/Ven based on clinical information before treatment. We utilized three clinical quantities for the analysis: Wilms Tumor-1 (WT1) mRNA expression level, the number of “favorable mutations”, and that of “unfavorable mutations”. Favorable and unfavorable mutations were defined as IDH1, IDH2, NPM1, and CEBPA mutations, and ASXL1, BCOR, EZH2, RUNX1, SF3B1, SRSF2, STAG2, U2AF1, ZRSR2 and TP53 mutations, respectively, based on the previous reports (NEJM 2019, Blood 2022). The data were standardized and utilized for the linear discriminant analysis. Statistical analyses were performed using R version 4.2.3 software (R Foundation for Statistical Computation, Vienna, Austria). [Results] Total 158 patients were enrolled in our study. Among 48 patients who received Aza/Ven as a 1 st line therapy, NGS analyses were performed prior to treatment in 38 patients. Their median age was 70.5 (39-92) years old. AML with myelodysplasia-related changes was the major subtype (71%) and 37 percent of the cases had high risk cytogenetic abnormalities at diagnosis. After 1 st line Aza/Ven treatment, complete remission (CR)/ CR with incomplete blood count recovery (CRi) was achieved in 63% of the cases while the remaining 37% were non-CR. There was a trend that patients with NPM1 and IDH1/2 mutations achieved CR/CRi, while patients with RUNX1 mutation were not tended to achieve CR (Figure 1). Next, we performed linear discriminant analysis of treatment response using three pretreatment clinical factors. Linear discriminant analysis found a linear combination of prognostic factors that best separates two classes in the group of patients. Thirty-seven patients were employed to determine the discriminant function D, given by D = 0.638 × x − 0.76 × y + 0.68 × z + 0.145. Here x, y, and z represented the WT1 quantity in peripheral blood, the number of “favorable mutations”, and that of “unfavorable mutations” after standardization, respectively. Using this model, 81.1% of cases were correctly classified, and so were 75.7% of cases with leave-one-out cross validation method (Table 1). [Discussions] As previously reported (NEJM 2019, Blood adv 2020), IDH1/2 and NPM1 mutations tended to be sensitive to Aza/Ven in our cohort. However, it would be hard to predict the response of Aza/Ven by a single gene mutation because multiple gene mutations were co-existed in most of the cases (Figure 1) and clinical factors including tumor markers might also affect the outcome. Our mathematical model, involving gene mutations and WT1, could efficiently predict the response of Aza/Ven, which may support the selection of 1 st line treatment. In conclusion, our study revealed the genetic landscape of real-world Aza/Ven therapy and provided a potential prognostic model.

Targeting cis-regulatory elements of FOXO family is a novel therapeutic strategy for induction of leukemia cell differentiation

Differentiation therapy has been proposed as a promising therapeutic strategy for acute myeloid leukemia (AML); thus, the development of more versatile methodologies that are applicable to a wide range of AML subtypes is desired. Although the FOXOs transcription factor represents a promising drug target for differentiation therapy, the efficacy of FOXO inhibitors is limited in vivo. Here, we show that pharmacological inhibition of a common cis-regulatory element of forkhead box O (FOXO) family members successfully induced cell differentiation in various AML cell lines. Through gene expression profiling and differentiation marker-based CRISPR/Cas9 screening, we identified TRIB1, a complement of the COP1 ubiquitin ligase complex, as a functional FOXO downstream gene maintaining an undifferentiated status. TRIB1 is direct target of FOXO3 and the FOXO-binding cis-regulatory element in the TRIB1 promoter, referred to as the FOXO-responsive element in the TRIB1 promoter (FRE-T), played a critical role in differentiation blockade. Thus, we designed a DNA-binding pharmacological inhibitor of the FOXO-FRE-T interface using pyrrole-imidazole polyamides (PIPs) that specifically bind to FRE-T (FRE-PIPs). The FRE-PIPs conjugated to chlorambucil (FRE-chb) inhibited transcription of TRIB1, causing differentiation in various AML cell lines. FRE-chb suppressed the formation of colonies derived from AML cell lines but not from normal counterparts. Administration of FRE-chb inhibited tumor progression in vivo without remarkable adverse effects. In conclusion, targeting cis-regulatory elements of the FOXO family is a promising therapeutic strategy that induces AML cell differentiation.

Myeloablative Dose of Busulfan and Fludarabine Combined with In Vivo T Cell Depletion Is Safe and Effective Conditioning for Acute Myeloid Leukemia and Myelodysplastic Syndrome Patients

Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative strategy for acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). The prediction of transplantation-related mortality (TRM) using the Hematopoietic Cell Transplantation Comorbidity Index (HCT-CI) score and an arbitrary upper age limit of 55 years for administering myeloablative conditioning (MAC) are common strategies to ensure a safe procedure. The use of reduced-toxicity conditioning regimens is an additional approach to providing safe and effective myeloablation. Herein we report the outcome of AML and MDS patients conditioned with fludarabine and a myeloablative dose of busulfan (FB4) stratified by age and HCT-CI score. The primary objective was overall survival (OS) for patients age ≥55 years. Secondary objectives were total OS, TRM, graft-versus-host disease (GVHD), and GVHD, relapse-free survival (GRFS). The 2 year OS was 72% in patients age <55 and 51% in patients age ≥55. In patients age ≥55 with an HCT-CI <2, the estimated 2 year OS was 64%, with median OS not reached. In those with HCT-CI ≥2, the 2-year OS was 43%, with a median OS of 14 months. The total cumulative incidence of relapse was 30% regardless of age or HCT-CI score. FB4 conditioning regimen offers a high rate of prolonged remission with a relapse rate similar to that reported in previous studies. These positive outcomes suggest that this conditioning platform can be offered to patients age ≥55 years in the absence of comorbidities, and that age should not be the sole determinant of conditioning intensity.

Poly ADP-ribose polymerase (PARP) inhibitors - new therapeutic strategies in Acute Myeloid Leukemia: a literature review

The use of poly (ADP-ribose) polymerase (PARP) inhibitors has recently increased as a result of demonstrating their superior efficacy compared to traditional chemotherapy in various cancer subtypes in many preclinical studies and clinical trials. A better understanding of the molecular mechanisms of PARP and their underlying foci that can be used as screening markers for potential new therapeutic options would aid rational treatment strategies and improve long-term patient outcomes. The available data on acute leukemia suggest potential windows for effective treatment of PARPi in disease subgroups. In this review, we summarize the current advances in the most common PARP-based adjuvant therapies and combination strategies for the treatment of acute leukemia and discuss the future prospects and challenges of therapy with PARP inhibitors. We also discuss reports describing an increased risk of cancer treatment in patients receiving PARP inhibitors for solid tumors.&#x0D; The research material were publications, the search was carried out using a combination of keywords such as: "PARP inhibitor", "acute leukemia", "therapy", "PARP clinical trials". The first step was to find relevant publications from the last 15 years. The second step was to review the publications found.&#x0D; Studies have shown that PARP inhibitors play an increasing role in the treatment of leukemia. In addition, focusing research efforts on identifying the most effective drug combinations and sequences could help to further shape the role of PARPi in the treatment of acute myeloid leukemia (AML).

A transient receptor potential channel-related model based on machine learning for evaluating tumor microenvironment and immunotherapeutic strategies in acute myeloid leukemia.

Acute myeloid leukemia (AML) is an aggressive hematopoietic malignancy. Transient receptor potential (TRP) channels in AML still need to be further explored. A TRP channel-related model based on machine learning was established in this study. The data were downloaded from TCGA-LAML and Genome-Tissue Expression (GTEx). TRP-related genes (TRGs) were extracted from previous literature. With the use of Single-Sample Gene Set Enrichment Analysis (ssGSEA), TRP enrichment scores (TESs) were calculated. The limma package was used to identify differentially expressed genes (DEGs), and univariate Cox regression analysis was performed to identify prognostic DEGs. The above prognostic DEGs were analyzed by Random Survival Forest and least absolute shrinkage and selection operator (Lasso) analysis to create the TRP signature. The Kaplan-Meier and receiver operating characteristic (ROC) curves were plotted to investigate the efficiency and accuracy of prognostic prediction. Moreover, genomic mutation analysis was based on GISTIC analysis. Based on ESTIMATE, TIMER, MCPcounter, and ssGSEA, the tumor microenvironment and immunological characteristics were expressly evaluated to explore immunotherapeutic strategies. Enrichment analysis for TRP signature was based on the Kyoto Encyclopedia of Genes Genomes (KEGG), Gene Ontology (GO), over-representation analysis (ORA), and Gene Set Enrichment Analysis (GSEA). Genomics of Drug Sensitivity in Cancer (GDSC) and pRRophetic were used to carry out drug sensitivity analysis. Conclusively, SCHIP1 was randomly selected to perform in vitro cyto-functional experiments. The worse clinical outcomes of patients with higher TESs were observed. There were 107 differentially expressed TRGs identified. Our data revealed 57 prognostic TRGs. Eight TRGs were obtained to establish the prognostic TRP signature, and the worse clinical outcomes of patients with higher TRP scores were found. The efficiency and accuracy of TRP signature in predicting prognosis were confirmed by ROC curves and five external validation datasets. Our data revealed that the mutation rates of DNMT3A, IDH2, MUC16, and TTN were relatively high. The level of infiltrating immune cell populations, stromal, immune, and ESTIMATE scores increased as the TRP scores increased. Nevertheless, AML patients with lower TRP scores exhibited more tumor purity. The TRP scores were found to be correlated with immunomodulators and immune checkpoints, thus revealing immune characteristics and immunotherapeutic strategies. The IC50 values of six chemotherapeutics were lower in the high TRP score (HTS) group. Finally, it was found that SCHIP1 may be the oncogenic gene. The results of this study will help in understanding the role of TRP and SCHIP1 in the prognosis and development of AML.

Breaking the Pump: Targeting the Sodium-Potassium Pump As a Therapeutic Strategy in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a heterogenous hematologic malignancy with poor overall survival despite intensive cytotoxic chemotherapy and newer targeted agents. With a 5-year survival rate of only 30% in adults with AML, new therapeutic strategies are needed. Tissue-specific differences in the expression of paralog genes, which are not essential in most cell types due to the buffering effect of the partner pair, could make for highly selective gene dependencies. To identify novel, selective paralogous targets for AML, we integrated the Cancer Dependency Map, which provides genome-scale CRISPR/Cas9 depletion screening data in hundreds of cancer cell lines, with numerous databases characterizing protein-protein interactions, paralog relationships, and gene expression in cancer models. Here, we identified ATP1B3 as a context-specific, paralog related dependency in AML. ATP1B3 is the smaller glycoprotein beta subunit of the sodium-potassium pump (Na/K-ATP pump). Together with the catalytic alpha subunit ATP1A1, it forms a heterodimer located in the plasma membrane, regulating the electrochemical gradient through the transport of Na and K ions across the membrane. The Na/K-ATP pump is of vital importance in the maintenance of cellular homeostasis and membrane potential. While ATP1A1 carries out the ion transport and enzymatic activity, making it a common essential gene (a gene which ranks among the most depleted genes in at least 90% of screened cell lines), ATP1B3 is not essential in most cancer cells. The beta subunit is important for intracellular transport and stabilization of the alpha subunit in the membrane, but it has three paralogs: ATP1B3 showing similar expression patterns across tissues, ATP1B2 exclusively expressed in brain and retina, and ATP1B1 showing differential expression across tissues. ATP1B1 has uniformly low expression in hematological malignancies and low ATP1B1 expression is the strongest predictor of ATP1B3 dependency (Figure 1). Using CRISPR/Cas9 knockout and competitive growth assays, we showed that knockout of ATP1B3 in ATP1B1 low-expressing AML cells indeed leads to synthetic lethality. Knockout of ATP1B3 significantly decreased number and size of colonies in a patient-derived xenograft (PDX) model of AML to a similar degree as the knockout of the common essential gene ATP1A1. Moreover, in these AML models, the absence of both paralogs of the beta subunit resulted in the destabilization of their binding partner ATP1A1. In contrast, expression of ATP1B1 stabilized ATP1A1 despite ATP1B3 knockout in solid tumors and in ATP1B1-overexpressing AML cells. In vivo, using an orthotopic AML cell line mouse model and an AML PDX model, ATP1B3 knockout resulted in lower leukemia burden in comparison to ATP1B1 knockout or a non-targeting control guide. To determine if ATP1B1 and ATP1B3 share primarily redundant functions, we utilized BioID (proximity-dependent biotin identification) to identify their interaction partners. We discovered that in AML, there is significant overlap of the ATP1B1 and ATP1B3 binding partners, and ATP1A1 is among the strongest. Enrichment of gene sets involved in protein folding in the endoplasmic reticulum are also consistent with the important role of the beta subunits in proper ATP1A1 folding and stabilization. Taken together, we identified ATP1B3 as a selective dependency in AML. We propose that when ATP1B1 is poorly expressed, elimination of ATP1B3 leads to the destabilization of the sodium-potassium pump including ATP1A1. ATP1B3 is a potential tumor-selective therapeutic target for AML and other hematologic malignancies with low expression of ATP1B1. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Allogeneic Hematopoietic Stem Cell Transplantation in Older Patients with Acute Myeloid Leukemia: Analysis of Outcomes and Contemporary Trends Spanning 10 Years

Background: Allogeneic hematopoietic stem cell transplantation (SCT) is a highly effective consolidation strategy in acute myeloid leukemia (AML). Non-myeloablative and reduced-intensity (NMA/RIC) conditioning regimens have expanded the use of SCT to older patients (pts) (age ≥ 60 years). Age alone no longer appears to be a barrier for successful SCT, with reported 3-year overall survival (OS) rates in the 45-50% range (Maakaron 2022). Methods: We conducted a retrospective study spanning 10 years to evaluate SCT-related outcomes in older AML. All pts ≥ 60 years presenting to our institution with newly-diagnosed (ND) AML were included. Acute promyelocytic leukemia (APL) was excluded. Median OS and relapse-free survival (RFS) were calculated using the Kaplan Meier method. A landmark analysis was performed to evaluate time-to-event endpoints in pts who underwent SCT in first response compared to those who did not. Results: Between 7/19/2012 and 11/17/2021, we identified 1073 pts ≥ 60 years with ND-AML. The median age at diagnosis was 71 years (range 60-94), 622 (58%) were male, 189 (18%) had prior myelodysplastic/myeloproliferative neoplasm, and 220 (21%) had therapy-related AML. ELN 2017 risk was favorable in 215 (20%), intermediate in 234 (22%), and adverse in 538 (50%) pts (86 with data missing). Most pts (84%) were treated with low-intensity (LOW) regimens. Only 16% were treated with intensive (INT) chemotherapy (intermediate or higher dose cytarabine). Venetoclax (VEN) was included in 34% of the regimens. The CR/CRi rate was 60% in the overall cohort. The CR/CRi rate was 74%, 48%, and 72% in pts treated with INT, LOW, and LOW + VEN therapies, respectively. The median OS and RFS were 11.2 and 10.6 months for the full cohort, respectively. The rate of referral to the SCT service was 38% (413/1073) and increased over time (31% in 2012-2013, 52% by 2020-2021). This occurred in parallel to increasing rates of CR/CRi (53% in 2012-2013, 66% by 2020-2021), likely reflecting increased efficacy of VEN-containing regimens in the later years. We identified 198/1073 pts (18%) who underwent SCT at some point during therapy, 152/198 (77%) in first response (CR/CRi/MLFS) after frontline therapy, and 46/198 (23%) following one or more salvage regimens. Of all ND-AML pts, the SCT rate was 37%, 10%, and 22% in pts treated with INT, LOW, and LOW + VEN, respectively. The rate of undergoing SCT increased over time (15% in 2012-2013, 27% by 2020-2021). Clinical/SCT characteristics for the pts who underwent SCT are shown in table 1. The rate of grade 3/4 acute graft-vs-host disease (GVHD) was 11% and 19% of pts experienced chronic GVHD of any grade. Following SCT, 24% of pts experienced relapse of AML and 21% died without disease relapse. The 100-day mortality following SCT was 13%. We then performed a landmark analysis (using median time to SCT [4.3 months] as the landmark) comparing pts undergoing SCT in first response following frontline therapy at our institution (n=139) to responding pts who did not receive SCT in first response and who were alive and without relapse at the landmark (n=353). Both the median OS (31.0 vs 18.9 months, p=0.0208, figure 1) and median RFS (23.2 vs 10.0 months, p<0.0001) were superior in the pts who underwent SCT compared to those who did not. Pts undergoing SCT in first response were younger (median age 66 vs 72 yrs) and had better performance status (PS 2-4 in 10% vs 22%) compared to no-SCT pts. Distribution of ELN risk categories and best response (CR/CRi/MLFS) were similar in the SCT and no-SCT groups. In pts who responded to initial therapy and were referred for SCT, but ultimately did not undergo SCT in first response (n=161), the most common documented reasons for forgoing SCT were social reasons (pt preference, financial/insurance/caregiver issues) in 29%, disease relapse in 29%, being deemed unfit due to comorbidities in 22%, active uncontrolled infections in 11%, and other reasons in 11%. An adequate donor was documented in 66% of these pts. Conclusions: Our results support the use of SCT as a consolidative strategy in older AML pts. Pts who undergo SCT have superior OS and RFS compared to those who do not. However, fewer than 20% of pts undergo the procedure mostly due to inadequate disease control, comorbidities, and a variety of social reasons. Recent trends indicate higher rates of SCT referral and completion as more effective anti-leukemic therapies are allowing a greater proportion of older AML pts to undergo SCT. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Imetelstat-Mediated Alterations in Lipid Metabolism to Induce Ferroptosis As Therapeutic Strategy for Acute Myeloid Leukemia

Imetelstat-Mediated Alterations in Lipid Metabolism to Induce Ferroptosis As Therapeutic Strategy for Acute Myeloid Leukemia

ME12 IDH Inhibitors in AML

IDH1 and IDH2 mutations occur in approximately 8-12% of patients with acute myeloid leukemia with some increase in frequency with age. Mutation of IDH converts alpha ketoglutarate into 2-hydroxy glutarate (2HG). 2HG has leukemogenic potential through epigenetic mechanisms. This has made targeting IDH mutations an attractive therapeutic strategy for acute myeloid leukemia. Enasidenib is a potent IDH2 inhibitor, the first drug of this class to be approved for treatment of patients with AML with IDH2 mutations.

S251: DISRUPTION OF SUCCINATE RECEPTOR SIGNALING PROMOTES MYELOPROLIFERATION AND ACUTE MYELOID LEUKAEMIA

Background: Unlike healthy haematopoietic stem cells (HSC), leukaemia stem cells (LSC) have high oxidative phosphorylation and hence targeting the tricarboxylic acid (TCA) cycle seems a promising therapeutic strategy in acute myeloid leukemia (AML) treatment. Accumulation of the TCA cycle oncometabolite succinate is associated with tumourigenesis, but its potential involvement in HSC function and leukaemia is unkown. Succinate stabilizes the oxygen-sensing transcription factor hypoxia-inducible factor (HIF)-1-α and activates interleukin (IL)-1β production in macrophages. Succinate may also be exported and has cell extrinsic effects through its receptor, succinate receptor-1 (SUCNR1), which may be pro- or anti-inflammatory. Aims: To investigate the in vivo role and molecular mechanisms of succinate and its receptor in normal and malignant haematopoiesis. Methods: To study the effect of succinate receptor on haematopoiesis, we characterized the immunophenotype of Sucnr1-KO mice. To dissect the role of Sucnr1 on healthy haematopoiesis via cell-autonomous effects, we transplanted fl/Sucnr1/fl control or Mx1-Cre fl/Sucnr1/fl bone marrow (BM) cells into wild-type (WT) C57BL/6J mice, which were later injected with pIpC. To study the role of Sucnr1 through non-cell autonomous effects, we transplanted WT BM into Nes-CreERT2fl/Sucnr1/fl recipients or fl/Sucnr1/fl controls, which were induced with tamoxifen. We characterized the effects of Sucnr1 deletion at the molecular level by bulk and single-cell RNA-sequencing (scRNA-seq), and aimed to uncover the molecular mechanisms mediating the effects of Sucnr1 ablation using in vivo tageting with drugs. To study the effect of succinate on leukaemic progression, we used two different mouse models, i.e. Mx1-Cre NrasG12D and MLL-AF9 knock-in (KI). In human AML, we quantified SUCNR1 expression in peripheral blood mononuclear cells (PBMC) from AML patients versus healthy volunteers, and treated xenograft recipients of CD34+ AML cells with succinate or vehicle. Results:In vivo deletion of Sucnr1 induced myeloid bias and expansion of the haematopoietic stem and progenitor cell (HSPC) compartment. In vivo deletion of Sucnr1 from Nestin+ stromal cells promoted expansion of long-term HSC (LT-HSC) versus intact recipients. Conversely, in vivo deletion of Sucnr1 from the haematopoietic system induced expansion of multipotent progenitors (MPP) versus intact haematopoietic cells. RNA-seq in HSPC from Sucnr1-KO versus WT mice showed changes in genes associated with myeloid output, and pinpointed related pathways potentially involved. scRNA-seq confirmed expansion of LT-HSC in Sucnr1-KO versus WT mice. In vivo treatments targeted signalling pathways at least partially responsible for the haematopoietic abnormalities in Sucnr1-KO. We found higher content of succinate in the BM extracellular fluid of leukaemic Mx1-Cre NrasG12D. Succinate injection to xenograft recipients of CD34+ AML cells increased leukaemic output versus vehicle, promoted myeloid bias in Mx1-Cre NrasG12D mice and reduced survival in mice transplanted with BM of MLL-AF9 leukaemic donors. PBMC from AML patients expressed lower level of SUCNR1 versus healthy donors and BM nucleated cells from Mx1-Cre NrasG12D also expressed less Sucnr1 than their healthy counterparts. Summary/Conclusion: We show a novel role for succinate and its receptor in the haematopoietic system and pinpoint this pathway as a novel means of communication within the BM HSC microenvironment, whose disruption may be involved in myeloid malignancy. These findings may help design new therapeutic strategies for AML patients.

Cyclin-dependent kinase 6 body methylation and association with prognosis of patients with acute myeloid leukemia.

e19000 Background: Cyclin-dependent kinase 6 (CDK6) plays critical roles in leukemogenesis and leukemia stem cells activation, and its inhibition has emerged as a novel therapeutic strategy in acute myeloid leukemia (AML) management. However, association between CDK6 methylation and prognosis in patients with AML remains unclear. Methods: Methylation profile, somatic alterations, and CDK6 gene expression data of 200 clinically annotated patients with de novo AML were retrieved from the TCGA Pan-Cancer Atlas project deposited in the UCSC Xena database. Specifically, methylation levels of all 64 CpGs across CDK6 were extracted, and then methylation abundances of promoter and gene body regions were calculated as average levels of CpGs located in the respective regions. Patients were categorized into two subgroups (hypo- and hyper-methylated) as per the median methylation level for either region or single CpG site. Log-rank tests were conducted to compare overall survival (OS) of patients stratified by methylation status. A Cox proportional hazards model was fitted to control confounding factors, including age, white blood cell count, cytogenetic risk classification (favorable, intermediate, and poor risk), and TP53 mutations, as reported previously. Associations of methylation status with cytogenetic risk and somatic alterations were examined using the Wilcoxon and Kruskal-Wallis rank sum tests, respectively. Spearman rank correlation coefficient ( R) was used to evaluate correlations between methylation and expression. Results: Of the 200 patients, 109 (54.5%) were men, and median (interquartile range [IQR]) age was 57.5 (44.75-67) years. Median (IQR) methylations of promoter and body were 5.2% (4.8%-5.7%) and 55.6% (53.7%-57.9%), respectively. Body methylation was observed to correlate with gene expression ( R, -0.22; P = .005), but not promoter methylation. In Kaplan-Meier analysis, low methylation of body rather than promoter predicted prolonged OS (median, 27.4 vs 9.2 months; hazard ratio [HR], 0.43; 95% CI, 0.29-0.63; P &lt; .001). Notably, the OS improvement persisted with multivariable Cox model adopted (HR, 0.64; 95% CI, 0.42-0.98; P = .038). In addition, the body hypomethylation was strongly associated with early age, favorable cytogenetic risk, mutations in TP53, NPM1, and IDH1/ 2, as well as PML- RARA and MYH11- CBFB fusions (all P &lt; .05). Further analysis identified that cg08300570, cg09653641, cg10481072, and cg13590558 in the body were all prognostic for OS (all P &lt; .05). Intriguingly, all these CpGs resides in the enhancer. Conclusions: This is the first study to unravel clinical significance of CDK6 body methylation in AML, and it may serve as an independent prognostic marker. Given that azacitidine and decitabine are widely used, our findings may have implications for combination therapy of CDK4/6 inhibitors with hypomethylating agents in AML. Prospective studies are warranted.

Inhibition of TKI-activated MIF/CXCR2 Pathway as a Novel Therapeutic Strategy in Acute Myeloid Leukemia

Abstract Despite significant advancements in our understanding of acute myeloid leukemia (AML), relapsed and refractory disease remains a major cause of treatment failure. Approximately 50% of patients with AML will develop relapsed disease following induction chemotherapy, which results in a dismal 5-year overall survival rate of 29%. The development of FMS-like tyrosine kinase 3 (FLT3) inhibitors have led to improved outcomes among patients with FLT3-mutated AML. However many patients eventually relapse and succumb to chemo-resistant disease, highlighting the need to characterize the molecular pathways which confer early TKI resistance. To explore the possible mechanisms responsible for the survival and proliferation of tyrosine kinase inhibitor (TKI) resistant blasts, we compared in vitro and ex vivo cytokine expression of gilteritinib (GILT)-treated and untreated blasts. Here we report that the pro-inflammatory cytokine macrophage migration inhibitory factor (MIF) is significantly expressed by GILT-treated blasts when compared to untreated controls. We further demonstrate that MIF expression promotes blast proliferation through the upregulation of its receptor CXCR2. Most remarkably we found that combination of CXCR2-inhibitor plus GILT works synergistically to reduce the percentage of viable blasts. Together these findings support that targeting the TKI-activated MIF/CXCR pathway could be a novel therapeutic strategy for both newly diagnosed and relapsed/refractory AML. This study is supported by Loma Linda University GRASP and Loma Linda University Research Innovation Grant