Acute Myeloid Leukemia Patient Specimens Research Articles

A Rapid Test to Evaluate the Tumor Differentiation State of Acute Myeloid Leukemias

Introduction: The combination of the hypomethylating agent Azacitidine with Venetoclax, a BCL2 inhibitor (Aza+Ven), is the frontline therapy for patients with acute myeloid leukemia (AML) who are elderly or unfit for intensive chemotherapy. However, identifying responsiveness to Aza+Ven a priori remains a clinical challenge.Aza+Ven effectively targets the primitive leukemia stem cell population (Pollyea, 2018), whereas resistance is observed in differentiated tumors (Pei, 2020). Further insights into the hierarchical organization of hematopoiesis have led to a definition of malignant AML cell types that follow the progression from hematopoietic stem cell to myeloid differentiated cells based on gene expression signatures, and these have been used to classify AML tumors (van Galen, 2019). Classification schemes using gene expression to capture variations in leukemic composition represent key biomarkers of response to targeted agents (Zeng, 2022; Bottomly, 2022). However, the prompt availability of nuanced cell state signatures to guide clinical decision making is limited by the laborious nature of performing and analyzing RNA-sequencing studies. Rapid AML stratification based on these classifications permits the possibility of identifying patients most likely to benefit from a drug or combination. Methods: To classify cell states based on transcriptomic signatures, we developed two assay formats, involving custom Nanostring and TaqMan Array card panels, for evaluation of tumor cell states for AML patients. Both formats contained gene sets representative of the six hematopoietic lineage states (van Galen, 2019) and both provide outputs that are compatible with the timeline for clinical decision-making. Assays were run according to the manufacturers' specifications, and enrichment of each of the six gene signatures was assessed using the Singscore stable method (Foroutan, 2018). Banked AML patient samples with matched RNA-seq and ex vivo Aza+Ven drug sensitivity data were used to validate the assay formats. Data obtained from the rapid assays were validated against cell states determined by deconvolution of bulk RNA sequence and compared with respect to clinical immunophenotype data where available. Results : Both the Nanostring and quantitative PCR-based assay formats detected gene expression representative of the appropriate cell state as previously determined by bulk RNA sequencing (Fig. 1A). Correlation of Aza+Ven ex vivo sensitivity with tumor cell state signatures revealed patterns where primitive but not differentiated cell states associated with sensitivity. In contrast, RNA isolated from healthy donor samples showed expression of all six cell state signatures. Across a cohort of 50 primary AML patient specimens, enrichment scores for each gene set correlated well with those derived for the same patients using deconvolution of bulk RNA-seq (Pearson r values: 0.785 to 0.944). Cell state characterization by rapid gene expression assays also aligned well with clinical immunophenotype, a routinely available diagnostic characterization of tumor blasts. Surface expression of primitive (CD117) and mature (CD11b) markers associated with Aza+Ven ex vivo sensitivity and resistance overall, respectively (p=0.03 & 0.04); however, ex vivo sensitivity could be significantly further stratified among samples expressing these markers when considering the gene expression-based enrichment scores for progenitor- and monocyte-like gene signatures (low vs high p-values: 0.02-0.05) (Fig. 1B). Analysis of matched samples from individual patients before and after treatment also showed cell state signature changes that reflected their temporal disease progression and correlated with differences in ex vivo drug sensitivity and clinical immunophenotype. Conclusion:Incorporation of cell state into prospective evaluations of AML patients provides an early indication of responsiveness to Aza+Ven. With rapid turnaround, assays such as these may augment treatment stratification and improve patient outcomes. These assays offer additional versatility to detect changes in AML cells after forced differentiation or acquired drug resistance.

Loss of CCAAT‐Enhancer Binding Protein Delta Promotes Acute Myeloid Leukemia Cell Proliferation and Survival By Upregulating Cyclin D1 Expression

Despite improvements in the treatment options, acute myeloid leukemia (AML) remains a fatal malignancy with an estimated 5-year relative survival rate of 31.7%. There is continued need for identification of new disease drivers to develop novel therapeutic options and improve clinical outcomes. Dysregulated transcriptional landscapes are associated with disease pathogenesis and treatment responses. To identify new disease drivers of AML, we analyzed RNA-Seq data from 59 patient-matched diagnosis and relapse (PAML) specimens (study cohort). A set of five differentially expressed genes (DEGs; STAT4, XRCC2, USP3, CEBPD, RBM47) with concordant changes in expression between diagnosis and relapse (Figure 1A) were selected for study using the following criteria: (a) concordant differential expression (DESeq2: log2FC > |1.2| and q < 0.05) in at least 25% of the relapsed patients in the study cohort (b) concordant differential expression in independent datasets of RNA-Seq without any experimental validation, and (c) high cancer driver potential based on their molecular function. Binding analysis for regulation of transcription (BART) on DEGs between normal CD34 + cells, diagnosis and relapsed AML patient specimens suggested that CEBPD could function as a transcriptional regulator of a subset of the DEGs. We hypothesized that dysregulated expression of these genes promotes AML disease progression. To examine their role in AML, we altered their expression in AML cell line models and measured changes in AML cell phenotypes. Of these genes, CRISPR-mediated knockdown of the transcription factor CCAAT/enhancer-binding protein delta (CEBPD) enhanced AML cell proliferation, self-renewal capacity, and survival of the relapsed AML cell line OCI-AML5. CEBPD knockdown also conferred OCI-AML5 resistance to cytarabine - a common chemotherapy drug used for AML treatment. Short hairpin RNA (shRNA)-mediated knockdown of CEBPD in OCI-AML5 validated these results. CRISPR-mediated upregulation of CEBPD expression induced monocyte/macrophage differentiation marker CD14 expression and enhanced apoptosis rates in OCI-AML5 cells. CEBPD upregulation also induced differentiation in the diagnosis AML cell line OCI-AML2. Survival analyses showed that lower expression of CEBPD was significantly associated with poor survival in pediatric AML patients, and there was a trend of lower CEBPD expression associating with worse survival in adult AML patients (UCSC Xena browser). These results altogether suggest that CEBPD plays a tumor suppressor role in AML. Gene set enrichment analyses (GSEA) on RNA-Seq data revealed significant positive enrichment of cell proliferation pathways upon CEBPD knockdown in OCI-AML5 cells as well as in a subset of the PAML patients with downregulated CEBPD expression at relapse. Differential gene expression and GSEA analyses altogether suggested upregulated cyclin D1 as one of the downstream drivers of enhanced growth of AML cells with repressed CEBPD expression (Figure 1B). RT-qPCR and Western Blot experiments confirmed upregulation of CCND1 expression in CEBPD knockdown OCI-AML5 and OCI-AML2 cells. Next, we sought to understand the molecular mechanisms repressing CEBPD expression during AML disease progression. We found an inverse association between CEBPD and DNMT3 methyltransferases mRNA expression in PAML and independent datasets. Treatment with the hypomethylating agent azacitidine upregulated CEBPD mRNA expression in multiple AML cell lines including OCI-AML5 and OCI-AML2, suggesting that hypermethylation represses CEBPD gene expression in these AML cells. Integrative analysis of gene expression with DNA methylation at the CEBPD promoter identified hypermethylation of multiple CpGs within the binding region of the transcription factor Sp1 in a subset of patients with CEBPD downregulation. CpG methylation has been shown to interfere with Sp1 binding at the CEBPD promoter. Since Sp1 binding promotes CEBPD transcription, hypermethylation at these sites may repress CEBPD expression during AML disease progression. Our study suggests that CEBPD can function as tumor suppressor in AML. Results suggest a role for DNA methylation in repressing CEBPD expression during AML disease progression. Furthermore, we show that CEBPD repression promotes AML cells' growth potentially via inducing the expression of the cell cycle driver CCND1.

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

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

Intracellular Calcium Localization Mediates the Activity of Venetoclax in Targeting Acute Myeloid Leukemia Stem Cells

Venetoclax (BCL2 inhibitor) based regimens have significantly improved outcomes for elderly patients with acute myeloid leukemia (AML). While most patients initially respond to venetoclax-based regimens, upfront resistance or relapse on therapy occurs in many. Therefore, elucidating the mechanistic link between BCL2 inhibition and AML cell death is crucial to finding novel therapeutic strategies to combat venetoclax resistance and to discover predictors of venetoclax response. Previous studies have shown BCL2 has a non-canonical function in modulating oxidative phosphorylation (OXPHOS) activity of AML stem cells (LSCs). However, the mechanistic link between BCL2 and OXPHOS has not yet been established. BCL2 has been shown to regulate intracellular calcium localization by modulating the activity of calcium channels at the ER and mitochondria. Optimal mitochondrial calcium concentrations are crucial for cell metabolism as calcium overload or decrease can inhibit OXPHOS activity. In preliminary studies, we established venetoclax increases mitochondrial calcium levels (Fig. 1A) leading to a subsequent decrease in OXPHOS activity and changes in the activity of key calcium dependent TCA cycle enzymes in primary human AML LSCs. Genetic inhibition of BCL2 in LSCs phenocopied the effects of venetoclax treatment. Based on these data, we hypothesized that BCL2 exerts its effect on LSC metabolism by modulating calcium localization between the ER and mitochondria. Furthermore, we sought to explore whether dysregulation of calcium localization by venetoclax is a mechanistic component of drug activity that leads to suppression of OXPHOS. To determine how BCL2 may be influencing mitochondrial calcium levels, we assessed the relationship between BCL2 and SERCA3, a key ion channel responsible for calcium uptake into the ER. Venetoclax disrupted the physical interaction between BCL2 and SERCA3. Mechanistically, venetoclax perturbed this interaction by decreasing SERCA3 protein levels. These findings were recapitulated by genetic inhibition of BCL2 in LSCs. Functional analysis of SERCA3 via siRNA-mediated knock-down in primary AML specimens demonstrated increased mitochondrial calcium levels, decreased OXPHOS activity and decreased engraftment of immune deficient NSG-S mice. Based on these data, we propose a model in which venetoclax disrupts SERCA function leading to less calcium uptake at the ER and more calcium sequestered into the mitochondria leading to overload, decreased OXPHOS activity and decreased LSC function. Next, we evaluated how calcium pathways were impacted in venetoclax resistant AML patient specimens. Intriguingly, venetoclax did not affect mitochondrial calcium levels (Fig. 1B) or the relationship between BCL2 and SERCA3. Therefore, we tested whether inhibiting the activity of MCU, the channel responsible for calcium uptake into the mitochondrial matrix, would target venetoclax resistant LSCs as it was overexpressed in this population. MCU inhibition led to decreased mitochondrial calcium levels, decreased OXPHOS activity and decreased LSC engraftment. These data indicate that venetoclax-resistant AML cells are no longer subject to changes in calcium localization induced by drug treatment but do retain an overall reliance on calcium regulation for LSC function/survival. Finally, we assessed whether our findings could be leveraged to predict venetoclax sensitivity. Analysis of single cell RNA-seq data from venetoclax sensitive versus resistant AML patient specimens (n=19) revealed multiple calcium pathway gene sets were enriched in venetoclax resistant LSCs. Additionally, levels of certain genes including SERCA3 and MCU were differentially expressed in primitive versus monocytic clusters between sensitive and resistant specimens. Taken together, our data demonstrate an unexpected and critical link between BCL2 and calcium signaling in human LSCs. A surprising aspect of our work is that venetoclax, a BH3 mimetic, acts to modulate the interaction with SERCA3. Lastly, our findings suggest that targeting intracellular calcium dynamics may represent a novel therapeutic strategy in the context of venetoclax resistant AML. In addition, we show that there are significant differences in calcium gene signatures between venetoclax sensitive vs resistant AML specimens that may be useful in predicting patient response to venetoclax based regimens. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The AP-1/ETS Transcription Factor Network Regulates Stem Cell Quiescence and Chemotherapy Resistance in Acute Myeloid Leukemia

Despite of the continuous improvements in the treatment for acute myeloid leukemia (AML), therapy resistance and disease relapse have been persistent and attributed to leukemia stem cells (LSCs). Recent single cell RNA sequencing (Galen et al, Bernstein 2019) and deconvoluted bulk RNA sequencing analysis (Zeng et al, Dick 2022) show hierarchical cellular organization in individual AML patient specimens, with LSCs, and quiescent LSCs in particular, at its apex. However, the mechanisms controlling LSCs and their quiescence remain elusive, hindering the development of effective targeted therapies. Here, using human patient AML specimens, we show that activator protein 1 (AP-1) and E26 transformation-specific (ETS) family transcription factors (TFs) coordinately regulate LSC quiescence and disease persistence after chemotherapy. A chemical label tracing technique using carboxyfluorescein succinimidyl ester (CFSE) has emerged as a powerful tool to identify quiescent LSCs, and using this technique, we and others have demonstrated that CFSE-label retaining quiescent LSCs isolated from patient leukemia cells exhibit potent leukemia initiating property, resistance to conventional chemotherapy, and epigenetic plasticity (Takao et al, Kentsis 2018 ASH, Ebinger et al, Jeremias 2016 and 2020). Here, we combined this method with functional genomic approaches to elucidate the mechanisms controlling quiescent LSCs and identify novel therapeutic targets to overcome chemotherapy resistance. First, to identify the candidate genes for LSC quiescence regulators, we performed gene expression and chromatin accessibility profiling of label-retaining quiescent LSCs isolated from human patient leukemia cell-engrafted NSG mice in diverse subtypes of AMLs using mRNA sequencing (RNA-seq) and assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq). We identified that several transcription factors important for normal hematopoiesis, which included AP-1 TFs, such as JUN and JUNB, and ETS TFs, such as ETS1 and FLI1, were differentially expressed in quiescent LSCs. Chromatin accessibility profiling further revealed the predominant occupancy of AP-1 and ETS TF family binding motifs in differentially accessible chromatin loci in quiescent LSCs. Importantly, gene expression signatures detected in quiescent LSCs showed significant similarity to gene expression profiles of residual leukemia cells observed in chemotherapy-treated AML patients (GSE116256, Galen et al. 2019), supporting the clinical relevance of our models. Next, to develop a functional screen system to identify the regulators of quiescent LSCs in human patient AML-bearing mice, we designed a lentiviral vector encoding mCherry fluorescent protein and doxycycline-inducible cDNAs marked by specific barcode sequences. cDNA library screening using this lentiviral vector identified several TFs as regulators for AML LSC quiescence, which included JUN as a positive regulator, and ETS1 and FLI1 as negative regulators (Figure 1). Finally, given the evidence of transcriptional activity of JUN to regulate LSC quiescence, we hypothesized that JUN activity may be also associated with chemotherapy resistance and disease persistence in patients. To investigate this, we analyzed JUN expression in individual AML cells in diverse patients before and after chemotherapy treatment (GSE116256, Galen et al. 2019). We found JUN to be highly expressed in persistent AML cells upon induction chemotherapy, especially in leukemia cells with features of hematopoietic stem cells (HSC-like), while ETS1 and FLI1 expression was not (Figure 2). These results argue that JUN activity may be required for chemotherapy resistance, LSC persistence, and ultimate AML relapse. In all, human AML LSC quiescence and therapy response can be dynamically regulated by a distinct transcription factor network. This work defines molecular mechanisms controlling AML LSC quiescence. We anticipate that these findings will lead to the elucidation of essential properties of LSC quiescence and the design of therapeutic strategies for their clinical identification and control. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Defining Clinical and Molecular Biomarkers for Venetoclax-Based Drug Combinations to Augment AML Therapy

Background: The combination of the BCL2 inhibitor venetoclax with azacytidine (Ven+Aza) is efficacious for elderly patients with acute myeloid leukemia (AML) who are unfit for standard chemotherapy. However, the overall and progression-free survival of these patients after 1 year of therapy is approximately 30-40%, and resistance to Ven+Aza, either primary or adaptive through clonal evolution, limits its durability. Therefore, we evaluated new venetoclax combinations to elucidate mechanisms and biomarkers of response and resistance. Methods: We tested a set of 31 venetoclax (Ven) combinations using an ex vivo assay on primary AML patient specimens (n=433, though not all combinations were screened for all samples). Enhanced efficacy was determined using a combination ratio (CR), defined as the sensitivity of the combination (by AUC) divided by the sensitivity of the most potent single agent AUC. Drug combination synergy was evaluated using the highest single agent (HSA) method. Clinical, genetic, and disease status information were collated, and ex vivo combination sensitivities were compared to matched single-agent data by Friedman test, across groups by Wilcoxon rank sum or Kruskal-Wallis test, and with continuous variables by Spearman rank correlation. For patient specimens with RNAseq data collected (n=279 patients), transcriptomic gene signatures of AML tumor cell state (van Galen, Cell 2019) were correlated with combination sensitivity by Spearman rank. Uni- and multi-variate analyses were applied to the full dataset to identify biomarkers of differential sensitivity to each combination. For one combination, Ven plus the JAK1/2 inhibitor ruxolitinib (Rux), additional genome wide CRISPR screens and follow up in vivo patient xenograft and mechanistic validation experiments using AML cell line models were performed. Results: Using Ven+Aza as a benchmark, we found several venetoclax combinations that exceeded both the ex vivo activity and enhanced efficacy observed with Ven+Aza in primary AML patient samples (Fig. 1A), while showing limited to no effect on stromal cell controls. Evaluation of clinical and genetic data with combination sensitivity revealed many significant transcriptomic, genetic, and immunophenotypic associations. The strong bias in sensitivity of Ven with respect to transcriptomic signatures of primitive tumor cell states compared to more mature, monocytic tumor states was unchanged with Ven+Aza and select other combinations. Importantly, this differential sensitivity was overcome when pairing Ven with several new drug partners such as Rux or the p38 MAPK inhibitor doramapimod (Fig. 1B). Uni- and multi-variate analyses further revealed an array of different biomarkers involving clinical and genetic features that correlated with significant differential sensitivity to each combination. Many of these likewise involved markers of cell differentiation state, such as CD14. To elucidate the mechanisms of enhanced efficacy of the Ven+Rux combination, which is currently under phase 1 clinical investigation, in vivo murine patient xenograft models and genome-wide CRISPR screens in OCI-AML2 cells were performed. Ven+Rux demonstrated superior inhibition of tumor growth in an AML xenograft model compared to either single-agent. The Ven+Rux CRISPR screen identified many genes whose knockdown was enriched in combination-resistant cells, including three (MDM2, MED13L, MEIS2) that were confirmed to confer resistance in subsequent immunoblotting and drug sensitivity validation experiments. In comparison, analysis of a control CRISPR screen using Ven alone revealed numerous gene hits in the JAK/STAT signaling pathway as enhancing the sensitivity of Ven. Conclusion: To expand the utility of venetoclax-inclusive combinations, we prospectively profiled a set of drug combinations and identified several with activity that exceeds Ven+Aza in ex vivo assays. Associations of clinical features with sensitivities to these drug combinations provide insight into their effectiveness which may be used to align the most promising combinations with translation to treatment of discrete subsets of AML patients. Together, these findings support opportunities for expanding the impact of Ven-based drug combinations in AML by leveraging clinical and molecular biomarkers of response. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

P482: NOVEL BISPECIFIC INNATE CELL ENGAGER AFM28 FOR THE TREATMENT OF CD123 POSITIVE ACUTE MYELOID LEUKEMIA AND MYELODYSPLASTIC SYNDROME

Background: Novel treatments for patients with acute myeloid leukemia (AML) and high-risk myelodysplastic syndrome (HR-MDS) with relapsed or refractory (R/R), or measurable residual disease are urgently needed. Targeting of the surface antigen CD123 expressed on both leukemic blasts and stem cells (LSCs) of most patients allows for depletion of both compartments, which is considered a prerequisite for deep anti-tumor responses and induction of prolonged remission. AFM28 is a novel bispecific Innate Cell Engager (ICE®) designed for bivalent high-affinity binding of CD16A on natural killer (NK) cells, redirecting effector cell cytotoxicity to CD123+ tumor cells. Allogeneic NK cell therapy is emerging as a next-generation treatment with demonstrated activity in R/R AML and a very benign safety profile. Retargeting of NK cells with AFM28 may be a particularly effective treatment strategy in patients with CD123+ AML and HR-MDS. Aims: 1. Preclinical characterization of AFM28 pharmacology and toxicology in vitro and in vivo to support clinical development. 2. Assessment of binding and activation of NK cells by AFM28 to test suitability for combination development with allogeneic NK cell therapy. Methods: Flow cytometry (FC) was used to assess binding and retention of AFM28 on NK cells, NK cell activation (degranulation, IFNγ expression) and depletion of leukemic cells from AML and MDS patient specimens. Quantification of secreted cytokines in leukocyte cultures employed bead-based luminex methodology. In vivo efficacy data were acquired by BLI measurement from a murine AML model. Cynomolgus data were acquired from non-GLP dose range finding and a GLP toxicology study using standard methods (ELISA, IHC, FC). Results: AFM28 induced NK cell activation at low picomolar concentrations and mediated efficacious depletion of CD123+ tumor cell lines and primary leukemic cells, including cells with low CD123 expression. NK cell activation was induced more potently than by an effector-enhanced IgG1 and was accompanied by more pronounced degranulation and IFNγ production. Similarly, AFM28 induced depletion of leukemic cells in patient bone marrow samples, without lysis of CD34+/CD123− cells, suggesting sparing of healthy hematopoietic progenitors. Potent anti-tumor activity was confirmed in vivo in a murine tumor model. In preclinical toxicology models, AFM28-induced target cell depletion was associated with minimal release of inflammatory cytokines, including IL-6, TNFα and IFNγ from leukocytes in vitro, and only low-level release of IL-6 in cynomolgus monkeys, despite efficacious depletion of CD123+ basophils and plasmacytoid dendritic cells (pDCs). Characterization of NK cell binding revealed high-avidity interaction, long cell surface retention, and potent induction of NK cell-mediated tumor cell lysis with no impact on NK cell viability. Summary/Conclusion: These data demonstrate that AFM28 exhibits potent pharmacological activity in vitro and in vivo and efficaciously induces NK cell-mediated depletion of primary leukemic cells. Further, toxicology data suggest good tolerability with a low risk of cytokine release syndrome. Together, these findings support the clinical investigation of AFM28 as a potential novel treatment of CD123+ AML and HR-MDS. A first-in-human trial investigating safety and activity of single agent AFM28 in patients with R/R AML is currently in preparation. In addition, these data support the combination of AFM28 with allogeneic NK cell therapy, which holds potential as an effective, novel treatment strategy.

RAS activation induces synthetic lethality of MEK inhibition with mitochondrial oxidative metabolism in acute myeloid leukemia

Despite recent advances in acute myeloid leukemia (AML) molecular characterization and targeted therapies, a majority of AML cases still lack therapeutically actionable targets. In 127 AML cases with unmet therapeutic needs, as defined by the exclusion of ELN favorable cases and of FLT3-ITD mutations, we identified 51 (40%) cases with alterations in RAS pathway genes (RAS+, mostly NF1, NRAS, KRAS, and PTPN11 genes). In 79 homogeneously treated AML patients from this cohort, RAS+ status were associated with higher white blood cell count, higher LDH, and reduced survival. In AML models of oncogenic addiction to RAS-MEK signaling, the MEK inhibitor trametinib demonstrated antileukemic activity in vitro and in vivo. However, the efficacy of trametinib was heterogeneous in ex vivo cultures of primary RAS+ AML patient specimens. From repurposing drug screens in RAS-activated AML cells, we identified pyrvinium pamoate, an anti-helminthic agent efficiently inhibiting the growth of RAS+ primary AML cells ex vivo, preferentially in trametinib-resistant PTPN11- or KRAS-mutated samples. Metabolic and genetic complementarity between trametinib and pyrvinium pamoate translated into anti-AML synergy in vitro. Moreover, this combination inhibited the propagation of RA+ AML cells in vivo in mice, indicating a potential for future clinical development of this strategy in AML.

Activation of Vitamin D Receptor Pathway Enhances Differentiating Capacity in Acute Myeloid Leukemia with Isocitrate Dehydrogenase Mutations.

Simple SummaryAround 15% of acute myeloid leukemia (AML) patients harbor mutations in isocitrate dehydrogenases (IDH), which lead to the production of the oncometabolite 2-hydroxyglutarate (2-HG). Inhibitors of mutant IDH enzymes and their 2-HG production have been approved by the FDA to be used in patients. However, 60% of IDH mutant AML patients do not respond to these inhibitors or develop mechanisms of resistance, leading to relapse. Among these mechanisms, some produce a 2-HG rebound. Alternative therapies exploiting the 2-HG-dependent molecular effects could therefore be of clinical interest. In this study, we demonstrate that 2-HG specifically activates vitamin D receptor (VDR) in IDH mutant AML cells leading to increased sensitivity to the combination of vitamin D (or VDR agonist) and all-trans retinoic acid and revealing a new therapeutic approach that can be readily applied to AML patients in this subgroup.Relapses and resistance to therapeutic agents are major barriers in the treatment of acute myeloid leukemia (AML) patients. These unfavorable outcomes emphasize the need for new strategies targeting drug-resistant cells. As IDH mutations are present in the preleukemic stem cells and systematically conserved at relapse, targeting IDH mutant cells could be essential to achieve a long-term remission in the IDH mutant AML subgroup. Here, using a panel of human AML cell lines and primary AML patient specimens harboring IDH mutations, we showed that the production of an oncometabolite (R)-2-HG by IDH mutant enzymes induces vitamin D receptor-related transcriptional changes, priming these AML cells to differentiate with pharmacological doses of ATRA and/or VD. This activation occurs in a CEBPα-dependent manner. Accordingly, our findings illuminate potent and cooperative effects of IDH mutations and the vitamin D receptor pathway on differentiation in AML, revealing a novel therapeutic approach easily transferable/immediately applicable to this subgroup of AML patients.

The ubiquitin ligase RNF5 determines acute myeloid leukemia growth and susceptibility to histone deacetylase inhibitors

Acute myeloid leukemia (AML) remains incurable, largely due to its resistance to conventional treatments. Here, we find that increased abundance of the ubiquitin ligase RNF5 contributes to AML development and survival. High RNF5 expression in AML patient specimens correlates with poor prognosis. RNF5 inhibition decreases AML cell growth in culture, in patient-derived xenograft (PDX) samples and in vivo, and delays development of MLL-AF9–driven leukemogenesis in mice, prolonging their survival. RNF5 inhibition causes transcriptional changes that overlap with those seen upon histone deacetylase (HDAC)1 inhibition. RNF5 induces the formation of K29 ubiquitin chains on the histone-binding protein RBBP4, promoting its recruitment to and subsequent epigenetic regulation of genes involved in AML maintenance. Correspondingly, RNF5 or RBBP4 knockdown enhances AML cell sensitivity to HDAC inhibitors. Notably, low expression of both RNF5 and HDAC coincides with a favorable prognosis. Our studies identify an ERAD-independent role for RNF5, demonstrating that its control of RBBP4 constitutes an epigenetic pathway that drives AML, and highlight RNF5/RBBP4 as markers useful to stratify patients for treatment with HDAC inhibitors.

LINC00987 knockdown inhibits the progression of acute myeloid leukemia by suppressing IGF2BP2-mediated PA2G4 expression.

This study aimed to investigate the role and potential mechanisms of LINC00987 in acute myeloid leukemia (AML) progression. The expression of LINC00987 in bone marrow specimens of AML patients and cell lines was measured by quantitative reverse transcription PCR (RT-qPCR). Small interfering RNA targeting LINC00987 (si-LINC00987) was transfected into AML cell lines HL-60 and KG-1, and the proliferation, invasion and apoptosis were detected with Cell Counting Kit-8 (CCK-8), Transwell and flow cytometry, respectively. Moreover, the binding between LINC00987 and insulin like growth factor 2 mRNA binding protein 2 (IGF2BP2) was validated with an RNA pull-down assay. Co-immunoprecipitation assay was used to verify the binding between IGF2BP2 and proliferation-associated 2G4 (PA2G4). Then rescue experiments were performed to explore the effects of LINC00987/IGF2BP2/PA2G4 axis on HL-60 and KG-1 cell functions. Additionally, HL-60 cells transfected with si-LINC00987 were injected into mice, followed by the evaluation of xenograft tumor growth. LINC00987 was upregulated in AML patient specimens and cell lines. LINC00987 knockdown inhibited proliferation and invasion and promoted apoptosis in AML cells. LINC00987 could bind with IGF2BP2 and promote its expression, and IGF2BP2 overexpression reversed the effects of LINC00987 knockdown on the proliferation, invasion and apoptosis in AML cells. Besides, IGF2BP2 could bind with PA2G4. IGF2BP2 knockdown inhibited proliferation and invasion, and promoted apoptosis in AML cells, whereas PA2G4 overexpression reversed these effects. Additionally, the LINC00987 knockdown inhibited the xenograft tumor growth of AML in vivo. Knockdown of LINC00987 inhibits AML cell proliferation and invasion, and promotes apoptosis in vitro and reduces tumor growth in vivo by suppressing IGF2BP2-mediated PA2G4 expression.

Abstract 1429: Characterization of trametinib combined with venetoclax in acute myeloid leukemia (AML) treatment

Abstract Acute myeloid leukemia (AML) is a type of hematologic malignancy with rapid progression and high genetic heterogeneity. Activating RAS pathway mutations are found in approximately 10 - 25% of AML patients and have been implicated to targeted drug resistance and disease relapse suggesting a potential benefit of MAPK pathway inhibitors in AML therapy. In patients with RAS-mutated myeloid malignancies, an inhibitor targeting MEK1/2, trametinib, demonstrated benefit in a phase 1/2 clinical study. Effective combinations of trametinib with other drugs has remained elusive. To identify potential combinatorial strategies, we generated AML cell lines resistant to MEKi. Whole exome sequencing revealed MEK1 variants in these cells. Drug sensitivity assays performed on parental AML cells and cells harboring wild type and the MEK1 variants validated their ability to confer trametinib resistance. BCL2 inhibitors, including venetoclax, were able to re-sensitize the resistant cells to trametinib, suggesting potential efficacy of combining trametinib with venetoclax. Indeed, this drug combination revealed synergistic efficacy in AML cell lines with diverse genetic backgrounds and in the resistant cells.To further investigate combination of trametinib and venetoclax in AML, we examined ~200 primary AML patient specimens using an ex vivo drug sensitivity assay, whole exome, and RNA sequencing data. We confirmed the enhanced efficacy of this combination relative to single agents. Patient samples resistant to trametinib showed increased sensitivity to venetoclax. Since, hyper-activation of MAPK signaling has been correlated with venetoclax resistance, we analyzed venetoclax resistant specimens and observed increased sensitivity to MEK and ERK inhibitors. We also observed correlation of MEK1 but not MEK2 RNA levels with trametinib sensitivity and negative correlation of MEK1 RNA levels with that of BCL2. AML samples with FLT3-ITD, NPM1, WT1, FLT3, CEBPA, DNMT3A, IDH1, IDH2, SRSF2, and TP53 mutations exhibited increased sensitivity to the combination. Samples with NRAS, KRAS, and PTPN11; although, highly sensitive to trametinib, did not benefit from this combination. Consistent with previous studies, we observed that venetoclax sensitivity was correlated with higher blast percentage. We also observed correlation of higher percentage of bone marrow blast and sensitivity to the combination. Moreover, while monocytic leukemias (AML-M4/M5) showed reduced sensitivity to venetoclax, they were sensitive to trametinib and showed increased expression of MEK1. Furthermore, we demonstrated that while venetoclax treatment increased protein levels of MCL1 in MOLM13 AML cells, combining trametinib with venetoclax reduced MCL1 levels. In summary, we demonstrate efficacy of trametinib and venetoclax combination in AML and propose mechanisms underlying the synergistic relationship of this drug combination. Citation Format: Alisa Damnernsawad, Christopher A. Eide, Sofia Beer, Haijiao Zhang, Daniel Bottomly, Stephen E. Kurtz, Jeffrey W. Tyner. Characterization of trametinib combined with venetoclax in acute myeloid leukemia (AML) treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1429.

Precision Targeting of Ion Channel Blocker for the Treatment of AML

The survival rate of patients with acute myeloid leukemia (AML) remains modest and thus, there is a clear need for advanced treatments. Here we describe two drug leads for AML that inhibit cellular sodium release without requiring drug internalization. EDC1 and EDC8 are two very potent extracellular antibody drug conjugates that selectively inhibit the all-important Na,K-ATPase, an ion pump required by cells to export excess sodium and to maintain ion gradients needed to import and export many important nutrients and waste products. We first demonstrate that dysadherin (a known metastatic cancer marker), is expressed on the surface many types of AML cell lines and can be used to direct Na,K-ATPase inhibitors using EDC1. We next demonstrate that ATRA inducible AML cells types are selectively destroyed with Na,K-ATPase inhibitors targeted to CD38 using EDC8 and ATRA.Since dysadherin expression is a known to be correlated to metastasis and stemness but yet to be studied in AML, we first analyzed a number of leukemia cell lines and AML patient samples for dysadherin expression by analyzing their surface expression and/or their sensitivity to EDC1. We observed that many cultured AML cells express dysadherin and are sensitive to EDC1 with EC50 values ranged from 0.02 to 1.0 nanoMolar and cell viability after 72 hours of exposure ranging from zero to 10%. For the three primary AML patient specimens analyzed, dysadherin expression was seen. Interestingly, in two of these three samples, the majority of intense staining was found in the CD123+ CD34+ population.Since CD38 expression and inducible expression can now be diagnosed in patients with AML, we analyzed if ATRA was required to sensitize AML to EDC8. Using multiple leukemic cell lines (HL60, U937, MV411, KG1, K562, and OCI/AML3) known to express CD38 with or without requiring ATRA, we analyzed EDC8 sensitivity before and after ATRA addition. We found that CD38 expression was required for EDC8 sensitivity and once expressed, EDC8 displayed potent activity with few viable cells detectable.Further studies analyzing dysadherin and CD38 expression in patient samples are needed to better understand the AML cell populations that may be affected by these EDCs but these results show the potential of this new type of antibody drug conjugate technology for the treatment of AML. EDCs that inhibit a required membrane bound ion pump with the precision of antibodies without requiring internalization may provide a new tool for doctors in their fight against AML. DisclosuresMarshall:CENTROSE: Employment. Malavasi:Jaansen Pharmaceuticals: Research Funding. Prudent:CENTROSE: Employment, Equity Ownership.

RNF5 Defines Acute Myeloid Leukemia Growth and Susceptibility to Histone Deacetylase Inhibitors

Acute myeloid leukemia (AML) remains an incurable blood cancer largely due to rapid emergence of resistance to conventional treatments. Thus, new therapeutic modalities are greatly needed to halt AML development. Here, using genetic and xenograft mouse models, we reveal that inhibition of the ubiquitin ligase RNF5 in human AML cell lines and in MLL-AF9-driven AML severely decreased the leukemogenic potential of those cells and prolonged survival of model leukemic mice. These findings suggest the possibility that targeting a single gene, namely RNF5, could effectively inhibit different AML subtypes. We initially focused on RNF5 as its expression is upregulated in AML patient cohorts as well as in AML-derived cell lines compared with normal hematopoietic cells. Furthermore, high RNF5 expression in AML patient specimens correlated with poor prognosis, relapse and short overall patient survival. By contrast, specimens from AML patients who responded to therapy exhibited low RNF5 levels. In vitro, RNF5 loss impaired the clonogenic potential of MLL-AF9-transduced bone marrow cells and markedly attenuated growth and survival of AML but not CML or T-ALL cell lines, in which RNF5 is also highly expressed. High-throughput screen and bioinformatics analysis identified RNF5 and ER-associated degradation (ERAD) components, as augmenting AML cell sensitivity to histone deacetylase (HDAC) inhibition. Indeed, inhibition of RNF5 sensitized AML cells to HDAC inhibitors. Correspondingly, a favorable prognosis was observed in AML patients exhibiting low expression of RNF5 and HDAC. Collectivity, our studies identify a potential new therapeutic modality based on targeting RNF5 to inhibit AML and suggest that RNF5 expression could serve as a prognostic marker and means to stratify patients for treatment with HDAC inhibitors. Disclosures Ofran: AbbVie: Membership on an entity's Board of Directors or advisory committees. Vuori:Bionano Genomics: Membership on an entity's Board of Directors or advisory committees.

Simultaneous kinase inhibition with ibrutinib and BCL2 inhibition with venetoclax offers a therapeutic strategy for acute myeloid leukemia.

Acute myeloid leukemia (AML) results from the enhanced proliferation and impaired differentiation of hematopoietic stem and progenitor cells. Using an ex vivo functional screening assay, we identified that the combination of the BTK inhibitor ibrutinib and BCL2 inhibitor venetoclax (IBR + VEN), currently in clinical trials for chronic lymphocytic leukemia (CLL), demonstrated enhanced efficacy on primary AML patient specimens, AML cell lines, and in a mouse xenograft model of AML. Expanded analyses among a large cohort of hematologic malignancies (n = 651 patients) revealed that IBR + VEN sensitivity associated with selected genetic and phenotypic features in both CLL and AML specimens. Among AML samples, 11q23 MLL rearrangements were highly sensitive to IBR + VEN. Analysis of differentially expressed genes with respect to IBR + VEN sensitivity indicated pathways preferentially enriched in patient samples with reduced ex vivo sensitivity, including IL-10 signaling. These findings suggest that IBR + VEN may represent an effective therapeutic option for patients with AML.

Anti-Apoptotic BCL-2 Family Members Confer Resistance to Calicheamicin-Based Antibody-Drug Conjugate Therapy of Acute Leukemia

BACKGROUND: With gemtuzumab ozogamicin (GO; targeting CD33) and inotuzumab ozogamicin (IO; targeting CD22), 2 antibody-drug conjugates delivering a toxic calicheamicin (CLM) derivative have recently been approved for the treatment of people with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), respectively. While effective in some, many patients do not benefit from these ADCs. It is unclear to what degree anti-apoptotic BCL-2 family members are involved in modulating efficacy of CLM-based ADCs, with limited studies coming to differing conclusions. Given the clinical availability of small molecule inhibitors for BCL-2 family proteins (BCLi), here we clarify the impact of BCL-2 family proteins on the anti-leukemic activity of CLM-ADCs. MATERIALS AND METHODS: Human AML and ALL cell lines were engineered to overexpress BCL-2, BCL-XL, and MCL-1 via lentiviral gene transfer. AML and ALL cell lines as well as AML patient samples were exposed to increasing concentrations of GO or IO with or without the BCL-2 inhibitor ABT-199 (venetoclax), the BCL-2/BCL-XL inhibitor ABT-263 (navitoclax), and the MCL-1 inhibitor AZD5991. Dead cells were enumerated by flow cytometry via 4',6-diamidino-2-phenylindole staining after 72 hours. For BH3 profiling of AML patient specimens, thawed AML patient specimen aliquots were exposed to JC-1 mitochondrial dye and BH3 peptides, and peptide-induced depolarization was then calculated as a percent relative to a CCCP positive control, yielding a priming score for each BH3 peptide. RESULTS: At a dose of 1000 pg/ml, GO killing of ML-1 (AML) cells decreased from 56±5% (mean±SEM) in parental cells to 32±7% (p<0.01) and 26±6% (p<0.01) in cells overexpressing BCL-2 and BCL-XL, respectively (all n=3). Similar results were seen in another AML cell line (HL-60). In REH (ALL) cells treated with IO, overexpression of BCL family members also reduced killing - at 500 pg/ml, 59±8% of cells were killed in contrast to 12±1% (p<0.01) of BCL-2-expressing and 11±1% (p<0.01) of BCL-XL-expressing cells, with similar results seen in another ALL cell line (RS4;11). Addition of ABT-199 or ABT-263 at 1 µM modestly increased GO-mediated killing of AML cell lines - for example, ML-1 cells treated with GO at 100 pg/ml, cytotoxicity increased from 41±6% to 57±7% (ABT-199, p<0.01) and 61±8% (ABT-263, p<0.01). The effect of BCLi was more pronounced on IO-mediated killing of ALL cell lines than on GO-mediated killing of AML lines. For example, killing of REH cells treated with IO at 25 pg/ml increased from 39±7% (without BCLi) to 72±8% (ABT-199 1 µM, p<0.01) and 87±9% (ABT-263 1 µM, p<0.01), with similar results seen in RS4;11 cells. BH3 peptide profiling of AML patient specimens treated with GO implicated MCL-1 as a potential additional modulator of AML response to GO. Consistent with this finding, overexpression of MCL-1 reduced leukemia cell death in HL-60 cells treated with GO (GO at 1000 pg/ml, 41±2 % vs. 26±1 %, p=0.01) and RS4;11 cells treated with IO (IO at 100 pg/ml, 76±2% vs. 27±6%, p<0.01). The MCL-1 inhibitor AZD5991 modestly increased the anti-leukemic efficacy of GO in ML-1 cells and AML patient specimens, but more dramatically enhanced IO killing of REH cells (IO at 10 pg/ml, 18±2% without AZD5991 vs. 70±2% with 0.1 µM AZD5991, p<0.01). The triplet combination of GO, ABT-199 and AZD5991 did not improve markedly on the ABT-199/AZD5991 combination in the absence of GO in cell lines or AML patient specimens, though the triplet combination of IO, ABT-199 and AZD5991 showed promising activity: in REH cells treated with 10 pg/ml IO, cytotoxicity was 18±2% without BCLi, 32±8% with ABT-199 0.1 µM, 19±2% with AZD5991 0.01 µM, and 56±14% with the triplet combination (p<0.01 for comparison of triplet combination with IO/BCLi doublet). CONCLUSIONS: Our studies establish an important role of anti-apoptotic BCL-2 family members as resistance factor for CLM-based ADC therapy of acute leukemia. These findings provide the rationale to explore the combination of small-molecule inhibitors of BCL-2 family members with CLM-ADCs as a combination strategy in the clinic to improve the efficacy of GO and, particularly, IO. These therapeutic strategies may incorporate the assessment of the relative contribution of specific BCL-2 family members to an individual cancer patient's disease. Disclosures Jean: Eutropics Pharmaceuticals: Employment. Cardone:Eutropics Pharmaceuticals: Employment, Equity Ownership. Walter:Seattle Genetics: Research Funding; Kite Pharma: Consultancy; Daiichi Sankyo: Consultancy; Jazz Pharmaceuticals: Consultancy; Agios: Consultancy; Amgen: Consultancy; Amphivena Therapeutics: Consultancy, Equity Ownership; Aptevo Therapeutics: Consultancy, Research Funding; Argenx BVBA: Consultancy; Astellas: Consultancy; BioLineRx: Consultancy; BiVictriX: Consultancy; Boehringer Ingelheim: Consultancy; Boston Biomedical: Consultancy; Covagen: Consultancy; New Link Genetics: Consultancy; Pfizer: Consultancy, Research Funding; Race Oncology: Consultancy.

Patterns of Sensitivity Exhibited By Venetoclax-Inclusive Drug Combinations in Acute Myeloid Leukemia

Patterns of Sensitivity Exhibited By Venetoclax-Inclusive Drug Combinations in Acute Myeloid Leukemia

Rspo-LGR4 Cooperates with HOXA9 to Sustain Self-Renewal in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is associated with high relapse rates and poor survival, with limited response to conventional cancer therapy and lacking effective targeting of highly self-renewing leukemic stem cells (LSCs). The mechanism underlying the high self-renewal activity of LSCs that determines the aggressiveness of disease remains poorly understood. Although we and others have previously demonstrated the clinical significance of aberrant WNT/β-catenin signaling in AML (Science, 327:1650-1653, 2010; Cancer Cell, 18:606-618, 2010), its pharmacologically tractable components essential for the regulation of LSC self-renewal have not yet been determined. Our studies discover, for the first time, a critical link between R-spondin (RSPO)-LGR4/HOXA9 and WNT/β-catenin pathways in AML LSCs. Microarray data analysis of 183 AML patient samples showed a significant positive correlation between expression of LGR4 and HOXA9 (r=0.546, P<0.0001). LGR4 exerted a cell-of-origin-specific function in promoting aberrant self-renewal and AML progression in vivo through cooperating with HOXA9, a poor prognostic predictor. We observed that LGR4 itself was not able to fully transform normal hematopoietic stem/progenitor cells (HSPCs), but instead cooperated with HOXA9 in HSPCs to accelerate disease onset producing a highly aggressive short latency AML in vivo. LGR4 and HOXA9 were epigenetically upregulated and their coexpression was an essential determinant of RSPO-LGR4 oncogenic activity. RSPO/WNT3 ligands could serve as stem cell growth factors to sustain myeloid differentiation block and to promote proliferation of CD34+ LSC-enriched subpopulations in primary AML patient specimens co-expressing LGR4 and HOXA9. Conversely, CRISPR/Cas9-mediated knockout of LGR4 not only suppressed RSPO/WNT3 signals and markedly decreased nuclear active β-catenin, but also reduced tumor burden in a patient-derived xenograft (PDX) mouse model of relapsed AML. Importantly, this study is the first to demonstrate that pharmacological inhibition of RSPO3-LGR4 signaling by a clinical-grade anti-RSPO3 monoclonal antibody induced LSC differentiation and consequently prevented tumor growth in AML PDX mice but did not affect normal human stem cell compartment in NSG mice. Together, our findings support a critical role for RSPO-LGR4 in the Wnt/β-catenin signaling pathway to promote AML leukemogenesis. Aberrant activation of RSPO-LGR4 is crucial for enhancing the self-renewal potential and myeloid differentiation block, which contribute to an aggressive leukemia phenotype through cooperating with HOXA9. Genetic and pharmacological targeting of this pathway impairs LSC self-renewal and survival and impedes AML development in murine models and patient-derived xenografts, highlighting the therapeutic value of targeting RSPO-LGR4 signaling in AML.

Long noncoding RNA MIAT promotes the progression of acute myeloid leukemia by negatively regulating miR-495

Acute myeloid leukemia (AML) is a malignant myeloid hematopoietic stem and progenitor cell disease. Studies have shown that the long noncoding RNA (lncRNA) myocardial infarction associated transcript (MIAT) is abundantly expressed in multiple human solid tumors. However, the expression and role of MIAT in AML has not been explored previously. In this study, we find that MIAT is overexpressed in AML patient specimens and AML cell lines. Importantly, upregulation of MIAT is closely related with poor clinical outcome. Further investigations reveal that knockdown of MIAT inhibits the colony formation and proliferation, meanwhile, accelerates the apoptosis of AML cells in vitro. Consistently, MIAT knockdown slows AML progression in immunodeficient mice. Mechanistically, we confirm that MIAT can function as a sponge to inhibit microRNA-495 (miR-495), a tumor suppressor, in AML cells. Collectively, our results demonstrate that MIAT is involved in promoting the progression of AML, at least partly, through negative regulation of miR-495, and therefore provide a promising target for treatment of AML.

Combining p38MAPK Inhibitors with a Second Targeted Agent Enhances Blockade of Inflammatory Signaling-Mediated Survival in Acute Myeloid Leukemia Cells

Combining p38MAPK Inhibitors with a Second Targeted Agent Enhances Blockade of Inflammatory Signaling-Mediated Survival in Acute Myeloid Leukemia Cells