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Acute myeloid leukemia (AML) is fatal in majority of adults. Identification of new therapeutic targets and their pharmacologic modulators are needed to improve outcomes. Previous studies had shown that immunization of rabbits with normal peripheral WBCs that had been incubated with fluorodinitrobenzene elicited high titer antibodies that bound to a spectrum of human leukemias. We report that proteomic analyses of immunoaffinity-purified lysates of primary AML cells showed enrichment of scaffolding protein IQGAP1. Immunohistochemistry and gene-expression analyses confirmed IQGAP1 mRNA overexpression in various cytogenetic subtypes of primary human AML compared to normal hematopoietic cells. shRNA knockdown of IQGAP1 blocked proliferation and clonogenicity of human leukemia cell-lines. To develop small molecules targeting IQGAP1 we performed in-silico screening of 212,966 compounds, selected 4 hits targeting IQGAP1-GRD domain, and conducted SAR of ‘fittest hit’ to identify UR778Br, a prototypical agent targeting IQGAP1. UR778Br inhibited proliferation, induced apoptosis, G2/M arrest, and colony formation by leukemia cell-lines and primary-AML while sparing normal marrow cells. IQGAP1/F-actin showed co-localization and UR778Br induced filopodia formation in U937 cells. UR778Br exhibited favorable ADME/T profiles and drug-likeness to treat AML. In summary, AML shows dependency on IQGAP1 and UR778Br, identified through in-silico studies, selectively targeted AML cells while sparing normal marrow.

Cell cycle analysis of acute leukemia cells treated with olaparib and AZD1775. A. The indicated cell lines were treated with DMSO (vehicle), olaparib (2 uM), and/or AZD1775 (200 nM) for 48 hr then fixed and stained with propidium iodide. Results are shown as mean {plus minus} SEM from a minimum of two independent experiments. B. .MV4;11 cells were treated with DMSO (vehicle), olaparib (2 uM), and/or AZD1775 (200 nM) for 48 hr then fixed and stained with propidium iodide. Representative plots are displayed. C. Jurkat cells were treated with DMSO (vehicle), olaparib (2 uM), and/or AZD1775 (200 nM) for 48 hr then fixed and stained with propidium iodide. Representative plots are displayed.

Venetoclax-based therapy can induce responses in approximately 70% of older previously untreated patients with acute myeloid leukemia (AML). However, up-front resistance as well as relapse following initial response demonstrates the need for a deeper understanding of resistance mechanisms. In the present study, we report that responses to venetoclax +azacitidine in patients with AML correlate closely with developmental stage, where phenotypically primitive AML is sensitive, but monocytic AML is more resistant. Mechanistically, resistant monocytic AML has a distinct transcriptomic profile, loses expression of venetoclax target BCL2, and relies on MCL1 to mediate oxidative phosphorylation and survival. This differential sensitivity drives a selective process in patients which favors the outgrowth of monocytic subpopulations at relapse. Based on these findings, we conclude that resistance to venetoclax + azacitidine can arise due to biological properties intrinsic to monocytic differentiation. We propose that optimal AML therapies should be designed so as to independently target AML subclones that may arise at differing stages of pathogenesis.Significance:Identifying characteristics of patients who respond poorly to venetoclax-based therapy and devising alternative therapeutic strategies for such patients are important topics in AML. We show that venetoclax resistance can arise due to intrinsic molecular/metabolic properties of monocytic AML cells and that such properties can potentially be targeted with alternative strategies.

Supplementary Figures S1-S5 supporting Main Figures 1-5.

PDF - 1495K, Quantification for the immunoblots shown in figure 5.

PDF - 3889K, AR-42 demonstrates higher activity against a panel of leukemia cell lines in contrast to PTL

PDF - 962K, Reactome gene list.

Most patients with acute myelogenous leukemia (AML) relapse and die of their disease. Increasing evidence indicates that AML relapse is driven by the inability to eradicate leukemia stem cells (LSC). Thus, it is imperative to identify novel therapies that can ablate LSCs. Using an in silico gene expression–based screen for compounds evoking transcriptional effects similar to the previously described anti-LSC agent parthenolide, we identified AR-42 (OSU-HDAC42), a novel histone deacetylase inhibitor that is structurally similar to phenylbutyrate, but with improved activity at submicromolar concentrations. Here, we report that AR-42 induces NF-κB inhibition, disrupts the ability of Hsp90 to stabilize its oncogenic clients, and causes potent and specific cell death of LSCs but not normal hematopoietic stem and progenitor cells. Unlike parthenolide, the caspase-dependent apoptosis caused by AR-42 occurs without activation of Nrf-2–driven cytoprotective pathways. As AR-42 is already being tested in early clinical trials, we expect that our results can be extended to the clinic. Mol Cancer Ther; 13(8); 1979–90. ©2014 AACR.

PDF - 306K, Differentially expressed genes upon AR-42 treatment.

PDF - 1783K, AR-42, unlike PTL, does not deplete thiols.

Supplementary figures and information

PDF - 67K, DAVID pathway analysis for downregulated genes (BBID).

Supplementary Tables S1-S6 describing patient characteristics, gene lists, LSC signatures, and reagents.

PDF - 3879K, AR-42 gene expression signature displays similarity to PTL and HDAC inhibitors.

Due to the disseminated nature of leukemia, malignant cells are exposed to many different tissue microenvironments, including a variety of extramedullary sites. In the present study, we demonstrate that leukemic cells residing in the liver display unique biological properties and also contribute to systemic changes that influence physiologic responses to chemotherapy. Specifically, the liver microenvironment induces metabolic adaptations via upregulating expression of endothelial lipase in leukemia cells, which not only stimulates tumor cell proliferation through polyunsaturated fatty acid–mediated pathways, but also promotes survival by stabilizing antiapoptotic proteins. Additionally, hepatic infiltration and tissue damage caused by malignant cells induces release of liver-derived enzymes capable of degrading chemotherapy drugs, an event that further protects leukemia cells from conventional therapies. Together, these studies demonstrate a unique role for liver in modulating the pathogenesis of leukemic disease and suggest that the hepatic microenvironment may protect leukemia cells from chemotherapeutic challenge.Significance:The studies presented herein demonstrate that the liver provides a microenvironment in which leukemia cells acquire unique metabolic properties. The adaptations that occur in the liver confer increased resistance to chemotherapy. Therefore, we propose that therapies designed to overcome liver-specific metabolic changes will yield improved outcomes for patients with leukemia.This article is highlighted in the In This Issue feature, p. 211

PDF - 77K, DAVID pathway analysis for downregulated genes (KEGG pathways).

PDF - 107K, AR-42, unlike PTL, does not upregulate HMOX-1 as a consequence of ROS induction.

Supplemental Data

Supplemental Tables 1-4. Table 1: pLKO shRNA clone information and antibody information. Table 2: Combination Indices to assess for synergism. Table 3: Quantitative 1H-, 31P- and 13C-NMR analysis of K562 CML cells treated with vehicle imatinib, oligomycin-A or the combination imatinib + oligomycin-A. Table 4: Complete blood counts and metabolic profiles after in vivo treatment with Oligomycin-A

Purpose:The prognosis of patients with multiple myeloma who are resistant to proteasome inhibitors, immunomodulatory drugs (IMiD), and daratumumab is extremely poor. Even B-cell maturation antigen–specific chimeric antigen receptor T-cell therapies provide only a temporary benefit before patients succumb to their disease. In this article, we interrogate the unique sensitivity of multiple myeloma cells to the alternative strategy of blocking protein translation with omacetaxine.Experimental Design:We determined protein translation levels (n = 17) and sensitivity to omacetaxine (n = 51) of primary multiple myeloma patient samples. Synergy was evaluated between omacetaxine and IMiDs in vitro, ex vivo, and in vivo. Underlying mechanism was investigated via proteomic analysis.Results:Almost universally, primary patient multiple myeloma cells exhibit >2.5-fold increased rates of protein translation compared with normal marrow cells. Ex vivo treatment with omacetaxine resulted in >50% reduction in viable multiple myeloma cells. In this cohort, high levels of translation serve as a biomarker for patient multiple myeloma cell sensitivity to omacetaxine. Unexpectedly, omacetaxine demonstrated synergy with IMiDs in multiple myeloma cell lines in vitro. In addition, in an IMiD-resistant relapsed patient sample, omacetaxine/IMiD combination treatment resensitized the multiple myeloma cells to the IMiD. Proteomic analysis found that the omacetaxine/IMiD combination treatment produced a double-hit on the IRF4/c-MYC pathway, which is critical to multiple myeloma survival.Conclusions:Overall, protein translation inhibitors represent a potential new drug class for myeloma treatment and provide a rationale for conducting clinical trials with omacetaxine alone and in combination with IMiDs for patients with relapsed/refractory multiple myeloma.

Purpose:Acute myeloid leukemia (AML) is a hematologic malignancy characterized by the accumulation of immature myeloid precursor cells. AML is poorly responsive to conventional chemotherapy and a diagnosis of AML is usually fatal. More effective and less toxic forms of therapy are desperately needed. AML cells are known to be highly dependent on the amino acid glutamine for their survival. These studies were directed at determining the effects of glutaminase inhibition on metabolism in AML and identifying general weaknesses that can be exploited therapeutically.Experimental Design:AML cancer cell lines, primary AML cells, and mouse models of AML and acute lymphoblastic leukemia (ALL) were utilized.Results:We show that blocking glutamine metabolism through the use of a glutaminase inhibitor (CB-839) significantly impairs antioxidant glutathione production in multiple types of AML, resulting in accretion of mitochondrial reactive oxygen species (mitoROS) and apoptotic cell death. Moreover, glutaminase inhibition makes AML cells susceptible to adjuvant drugs that further perturb mitochondrial redox state, such as arsenic trioxide (ATO) and homoharringtonine (HHT). Indeed, the combination of ATO or HHT with CB-839 exacerbates mitoROS and apoptosis, and leads to more complete cell death in AML cell lines, primary AML patient samples, and in vivo using mouse models of AML. In addition, these redox-targeted combination therapies are effective in eradicating ALL cells in vitro and in vivo.Conclusions:Targeting glutamine metabolism in combination with drugs that perturb mitochondrial redox state represents an effective and potentially widely applicable therapeutic strategy for treating multiple types of leukemia.

Supplemental Figures 1-8. Figure 1. Mitochondrial metabolism becomes essential for TKI-treated BCR-ABL+ leukemia cells. Figure 2: Oligomycin-A sensitizes cells to BCR-ABL inhibition. Figure 3. Oligomycin-A sensitizes cells to BCR-ABL inhibition. Figure 4. Oligomycin-A sensitizes acute myeloid leukemia cells to TKI. Figure 5. Low nM concentrations of oligomycin-A do not perturb the TCA cycle. Figure 6. Oligomycin-A disrupts mitochondrial functions in leukemia cells. Figure 7. Low dose oligomycin-A synergizes with TKI to eliminate leukemia in vivo with no significant toxicity. Figure 8. Model for how TKI treatment of leukemias creates an altered metabolic state sensitive to mitochondrial perturbations.

Supplemental Methods. This supplementary file includes additional methods for sample preparation, Seahorse analysis of oxygen consumption rates, reactive oxygen species measurements, flow cytometry analysis, NMR analysis, RT-PCR conditions, quizartinib synthesis information.

Figure S1. The glutaminase inhibitor CB-839 impairs glutathione metabolism in FLT3WT AML cells. Figure S2. The glutaminase inhibitor CB-839 decreases glutathione levels, but does not increase total cellular ROS levels in AML cells. Figure S3. Antioxidant vitamin E suppresses mitoROS and apoptosis induced by CB-839/pro-oxidant combination therapies. Figure S4. CB-839 cooperates with the pro-oxidant drug ATO in inducing apoptosis, mitoROS and AML cell death. Figure S5. CB-839/HHT therapy induces total cellular ROS. AML cells that survive therapy are metabolically similar to drug naive cells and are not resistant to subsequent therapy. Figure S6. CB-839/ATO and CB-839/HHT combination therapies do not exhibit toxicity in vivo in mice or toward normal human CD34+ cells. Figure S7. CB-839 cooperates with HHT in inducing mitoROS and apoptosis in primary human AML cells and cell death/apoptosis in ALL cells. Table S1. Combination index (CI values) for drug combinations tested in cell viability assays. Table S2. The clinical characteristics of the AML cohort studied are summarized.

Purpose: Although tyrosine kinase inhibitors (TKI) can be effective therapies for leukemia, they fail to fully eliminate leukemic cells and achieve durable remissions for many patients with advanced BCR-ABL+ leukemias or acute myelogenous leukemia (AML). Through a large-scale synthetic lethal RNAi screen, we identified pyruvate dehydrogenase, the limiting enzyme for pyruvate entry into the mitochondrial tricarboxylic acid cycle, as critical for the survival of chronic myelogenous leukemia (CML) cells upon BCR-ABL inhibition. Here, we examined the role of mitochondrial metabolism in the survival of Ph+ leukemia and AML upon TK inhibition.Experimental Design: Ph+ cancer cell lines, AML cell lines, leukemia xenografts, cord blood, and patient samples were examined.Results: We showed that the mitochondrial ATP-synthase inhibitor oligomycin-A greatly sensitized leukemia cells to TKI in vitro. Surprisingly, oligomycin-A sensitized leukemia cells to BCR-ABL inhibition at concentrations of 100- to 1,000-fold below those required for inhibition of respiration. Oligomycin-A treatment rapidly led to mitochondrial membrane depolarization and reduced ATP levels, and promoted superoxide production and leukemia cell apoptosis when combined with TKI. Importantly, oligomycin-A enhanced elimination of BCR-ABL+ leukemia cells by TKI in a mouse model and in primary blast crisis CML samples. Moreover, oligomycin-A also greatly potentiated the elimination of FLT3-dependent AML cells when combined with an FLT3 TKI, both in vitro and in vivo.Conclusions: TKI therapy in leukemia cells creates a novel metabolic state that is highly sensitive to particular mitochondrial perturbations. Targeting mitochondrial metabolism as an adjuvant therapy could therefore improve therapeutic responses to TKI for patients with BCR-ABL+ and FLT3ITD leukemias. Clin Cancer Res; 21(6); 1360–72. ©2014 AACR.

PDF file - 772KB, Supplementary Figure 1. Selenium supplementation abrogates leukemia in a Friend leukemia (FVP) model. Supplementary Figure 2. Effect of selenium supplementation on the splenic histopathology in FVP-infected mice. Supplementary Figure 3. Selenium supplementation increases apoptosis in the spleen of FVP infected mice.Supplementary Figure 4. Essential role of the COX/eicosanoid pathway in the ablation of LSCs in FVP-infected Se-S mice. Supplementary Figure 5. Modulation of expression of NADPH oxidases (A) and Gpx4 (B) by selenium-treatment of BCR-ABL+LSCs. Supplementary Figure 6. Expression of Tp53, Cat and Sod2 in the spleen of FVP-infected mice. Supplementary Figure 7. Activation of p53 in FVP-infected Se-S and MSA mice. Supplementary Figure 8. Selenium-dependent expression of selenoproteins and CyPG synthesis machinery and endogenous production of 12-PGJ2 by LC-MS/MS in BCRABL+ LSCs.

Key Points Patients with AML who received IC were compared with those who received ven to investigate predictors of outcomes.Presence of RUNX1 mutations is associated with better outcomes for ven/aza compared with IC., Visual Abstract, Venetoclax (ven) plus azacitidine (aza) is the standard of care for patients with newly diagnosed acute myeloid leukemia (AML) who are not candidates for intensive chemotherapy (IC). Some patients who are IC candidates instead receive ven/aza. We retrospectively analyzed patients with newly diagnosed AML who received ven/aza (n = 143) or IC (n = 149) to compare outcomes, seek variables that could predict response to 1 therapy or the other, and ascertain whether treatment recommendations could be refined. The response rates were 76.9% for ven/aza and 70.5% for IC. The median overall survival (OS) was 884 days for IC compared with 483 days for ven/aza (P = .0020). A propensity-matched cohort was used to compare outcomes in the setting of equivalent baseline variables, and when matched for age, biological risk, and transplantation, the median OS was 705 days for IC compared with not reached for ven/aza (P = .0667). Variables that favored response to ven/aza over IC included older age, secondary AML, and RUNX1 mutations. AML M5 favored response to IC over ven/aza. In the propensity-matched cohort analyzing OS, older age, adverse risk, and RUNX1 mutations favored ven/aza over IC, whereas intermediate risk favored IC over ven/aza. In conclusion, patients receiving IC have improved OS compared with those receiving ven/aza. However, in a propensity-matched cohort of patients with equivalent baseline factors, there was a trend toward favorable OS for ven/aza. Specific variables, such as RUNX1 mutations, reported here for the first time, can be identified that favor ven/aza or IC, helping to guide treatment decisions for patients who may be eligible candidates for either therapy.

The BCL-2 inhibitor venetoclax has recently emerged as an important component of acute myeloid leukemia (AML) therapy. Notably, use of this agent has revealed a previously unrecognized form of pathogenesis characterized by monocytic disease progression. We demonstrate that this form of disease arises from a fundamentally different type of leukemia stem cell (LSC), which we designate as monocytic LSC (m-LSC), that is developmentally and clinically distinct from the more well-described primitive LSC (p-LSC). The m-LSC is distinguished by a unique immunophenotype (CD34-, CD4+, CD11b-, CD14-, CD36-), unique transcriptional state, reliance on purine/pyrimidine metabolism, and selective sensitivity to cladribine. Critically, in some instances m-LSC and p-LSC subtypes can co-reside in the same AML patient and simultaneously contribute to overall tumor complexity. Thus, our findings demonstrate that LSC heterogeneity has direct clinical significance and highlights the need to distinguish and target m-LSCs as a means to improve clinical outcomes with venetoclax-based regimens.Statement of SignificanceThese studies identify and characterize a new type of human acute myeloid leukemia stem cell (LSC) that is responsible for monocytic disease progression in acute myeloid leukemia (AML) patients treated with venetoclax-based regimens. Our studies describe the phenotype, molecular properties, and drug sensitivities of this unique LSC subclass.

The combination of venetoclax (ven) and azacitidine (aza) has resulted in high response rates in the upfront treatment of AML in patients age > 75 and patients unfit for intensive chemotherapy. Given the poor historical outcomes in patients age ≥ 60 treated with induction chemotherapy, ven/aza has become our institutional preference for the initial treatment of non-core binding factor (CBF) AML patients age ≥ 60. The benefit of allogeneic stem cell transplant (SCT) in patients who achieve response to ven/aza is uncertain. We report outcomes of SCT-eligible patients treated at our center. Between 1/2015 and 1/2020, 119 newly diagnosed non-CBF AML patients age ≥ 60 received ven/aza as initial therapy. 21 patients underwent SCT; 31 additional patients were potentially SCT eligible but deferred SCT. Overall survival (OS) was significantly greater among SCT patients (median survival not reached) versus potentially SCT eligible patients not undergoing SCT (median 518 days) (p = 0.01). Our data suggest that ven/aza followed by SCT in newly diagnosed AML patients older than ≥ 60 results in excellent outcomes and likely improves outcomes over maintenance therapy. Ongoing investigation will further refine the optimal timing of and selection of patients for SCT based on prognostic disease features and response assessments.

Understanding the genetic underpinnings and clonal structure of malignancies at single-cell resolution is critical to accurately predicting drug response and understanding mechanisms of drug resistance and disease evolution in heterogeneous populations of cells. Here, we introduce an accessible, multiplexable, targeted mutation enrichment approach and end-to-end analysis pipeline called MuVEH (Multiplexed Variant Enrichment by Hybridization) that increases the resolution of variant detection in scRNA-seq analysis. When applied specifically to the mitochondrial chromosome (“mitoMuVEH”), this technique can also be used to reconstruct and trace clonal relationships between individual cells. We applied both approaches to two pairs of primary bone marrow specimens from acute myelogenous leukemia (AML) patients collected at diagnosis and after relapse following Venetoclax+Azacitidine (Ven/Aza) therapy. Used together, MuVEH and mitoMuVEH reveal clonal evolution and changing mutational burden in response to treatment at single-cell resolution in these patients. Ultimately, these approaches have the potential to extract additional biological insights from precious patient samples and provide insight into the contributions clonality and genotype have during disease progression.

Alternative cleavage and polyadenylation (APA) contributes to the diversity of mRNA 3′ ends, affecting post-transcriptional regulation by including or excluding cis-regulatory elements in mRNAs, altering their stability and translational efficiency. While APA analysis has been applied broadly in mixed populations of cells, the heterogeneity of APA among single cells has only recently begun to be explored. We developed an approach we termed scraps (Single Cell RNAPolyA Site Discovery), implemented as a user-friendly, scalable, and reproducible end-to-end workflow, to identify polyadenylation sites at near-nucleotide resolution in single cells using 10X Genomics and other TVN-primed single-cell RNA-seq (scRNA-seq) libraries. Our approach, which performs best with long (>100bp) read 1 sequencing and paired alignment to the genome, is both unbiased relative to existing methods that utilize only read 2 and recovers more sites at higher resolution, despite the reduction in read quality observed on most modern DNA sequencers following homopolymer stretches. For libraries sequenced without long read 1, we implement a fallback approach using read 2-only alignments that performs similarly to our optimal approach, but recovers far fewer polyadenylation sites per experiment. scraps also enables assessment of internal priming capture events, which we demonstrate occur commonly but at higher frequency during apoptotic 3′ RNA decay. We also provide an R package, scrapR, that integrates the results of the scaps pipeline with the popular Seruat single-cell analysis package. Refinement and expanded application of these approaches will further clarify the role of APA in single cells, as well as the effects of internal priming on expression measurements in scRNA-seq libraries.

The plant-derived sesquiterpene lactone micheliolide was recently found to possess promising antileukemic activity, including the ability to target and kill leukemia stem cells. Efforts toward improving the biological activity of micheliolide and investigating its mechanism of action have been hindered by the paucity of preexisting functional groups amenable for late-stage derivatization of this molecule. Here, we report the implementation of a probe-based P450 fingerprinting strategy to rapidly evolve engineered P450 catalysts useful for the regio- and stereoselective hydroxylation of micheliolide at two previously inaccessible aliphatic positions in this complex natural product. Via P450-mediated chemoenzymatic synthesis, a broad panel of novel micheliolide analogs could thus be obtained to gain structure–activity insights into the effect of C2, C4, and C14 substitutions on the antileukemic activity of micheliolide, ultimately leading to the discovery of “micheliologs” with improved potency against acute myelogenic leukemia cells. These late-stage C–H functionalization routes could be further leveraged to generate a panel of affinity probes for conducting a comprehensive analysis of the protein targeting profile of micheliolide in leukemia cells via chemical proteomics analyses. These studies introduce new micheliolide-based antileukemic agents and shed new light onto the biomolecular targets and mechanism of action of micheliolide in leukemia cells. More broadly, this work showcases the value of the present P450-mediated C–H functionalization strategy for streamlining the late-stage diversification and elucidation of the biomolecular targets of a complex bioactive molecule., A P450-based chemoenzymatic C−H functionalization strategy was applied to discover improved analogs of the anticancer terpene micheliolide and to profile its target proteome in leukemia stem cells.