Mohammad Minhajuddin

Targeting Aberrant Glutathione Metabolism to Eradicate Human Acute Myelogenous Leukemia Cells

Background: Eradication of primary human leukemia cells represents a major challenge. Therapies have not substantially changed in over 30 years. Results: Using normal versus leukemia specimens enriched for primitive cells, we document aberrant regulation of glutathione metabolism. Conclusion: Aberrant glutathione metabolism is an intrinsic property of human leukemia cells. Significance: Interventions based on modulation of glutathione metabolism represent a powerful means to improve therapy. The development of strategies to eradicate primary human acute myelogenous leukemia (AML) cells is a major challenge to the leukemia research field. In particular, primitive leukemia cells, often termed leukemia stem cells, are typically refractory to many forms of therapy. To investigate improved strategies for targeting of human AML cells we compared the molecular mechanisms regulating oxidative state in primitive (CD34+) leukemic versus normal specimens. Our data indicate that CD34+ AML cells have elevated expression of multiple glutathione pathway regulatory proteins, presumably as a mechanism to compensate for increased oxidative stress in leukemic cells. Consistent with this observation, CD34+ AML cells have lower levels of reduced glutathione and increased levels of oxidized glutathione compared with normal CD34+ cells. These findings led us to hypothesize that AML cells will be hypersensitive to inhibition of glutathione metabolism. To test this premise, we identified compounds such as parthenolide (PTL) or piperlongumine that induce almost complete glutathione depletion and severe cell death in CD34+ AML cells. Importantly, these compounds only induce limited and transient glutathione depletion as well as significantly less toxicity in normal CD34+ cells. We further determined that PTL perturbs glutathione homeostasis by a multifactorial mechanism, which includes inhibiting key glutathione metabolic enzymes (GCLC and GPX1), as well as direct depletion of glutathione. These findings demonstrate that primitive leukemia cells are uniquely sensitive to agents that target aberrant glutathione metabolism, an intrinsic property of primary human AML cells.

Leukemic Stem Cells Evade Chemotherapy by Metabolic Adaptation to an Adipose Tissue Niche

Adipose tissue (AT) has previously been identified as an extra-medullary reservoir for normal hematopoietic stem cells (HSCs) and may promote tumor development. Here, we show that a subpopulation of leukemic stem cells (LSCs) can utilize gonadal adipose tissue (GAT) as a niche to support their metabolism and evade chemotherapy. In a mouse model of blast crisis chronic myeloid leukemia (CML), adipose-resident LSCs exhibit a pro-inflammatory phenotype and induce lipolysis in GAT. GAT lipolysis fuels fatty acid oxidation in LSCs, especially within a subpopulation expressing the fatty acid transporter CD36. CD36(+) LSCs have unique metabolic properties, are strikingly enriched in AT, and are protected from chemotherapy by the GAT microenvironment. CD36 also marks a fraction of human blast crisis CML and acute myeloid leukemia (AML) cells with similar biological properties. These findings suggest striking interplay between leukemic cells and AT to create a unique microenvironment that supports the metabolic demands and survival of a distinct LSC subpopulation.

Chemical genomic screening reveals synergism between parthenolide and inhibitors of the PI-3 kinase and mTOR pathways

We have previously shown that the plant-derived compound parthenolide (PTL) can impair the survival and leukemogenic activity of primary human acute myeloid leukemia (AML) stem cells. However, despite the activity of this agent, PTL also induces cellular protective responses that likely function to reduce its overall cytotoxicity. Thus, we sought to identify pharmacologic agents that enhance the antileukemic potential of PTL. Toward this goal, we used the gene expression signature of PTL to identify compounds that inhibit cytoprotective responses by performing chemical genomic screening of the Connectivity Map database. This screen identified compounds acting along the phosphatidylinositol 3-kinase and mammalian target of rapamycin pathways. Compared with single agent treatment, exposure of AML cells to the combination of PTL and phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitors significantly decreased viability of AML cells and reduced tumor burden in vitro and in murine xenotransplantation models. Taken together, our data show that rational drug combinations can be identified using chemical genomic screening strategies and that inhibition of cytoprotective functions can enhance the eradication of primary human AML cells.

Rational Design of a Parthenolide-based Drug Regimen that Selectively Eradicates Acute Myelogenous Leukemia Stem Cells.

Although multidrug approaches to cancer therapy are common, few strategies are based on rigorous scientific principles. Rather, drug combinations are largely dictated by empirical or clinical parameters. In the present study we developed a strategy for rational design of a regimen that selectively targets human acute myelogenous leukemia (AML) stem cells. As a starting point, we used parthenolide, an agent shown to target critical mechanisms of redox balance in primary AML cells. Next, using proteomic, genomic, and metabolomic methods, we determined that treatment with parthenolide leads to induction of compensatory mechanisms that include up-regulated NADPH production via the pentose phosphate pathway as well as activation of the Nrf2-mediated oxidative stress response pathway. Using this knowledge we identified 2-deoxyglucose and temsirolimus as agents that can be added to a parthenolide regimen as a means to inhibit such compensatory events and thereby further enhance eradication of AML cells. We demonstrate that the parthenolide, 2-deoxyglucose, temsirolimus (termed PDT) regimen is a potent means of targeting AML stem cells but has little to no effect on normal stem cells. Taken together our findings illustrate a comprehensive approach to designing combination anticancer drug regimens.

Flavaglines target primitive leukemia cells and enhance anti-leukemia drug activity

Identification of agents that target human leukemia stem cells is an important consideration for the development of new therapies. The present study demonstrates that rocaglamide and silvestrol, closely related natural products from the flavagline class of compounds, are able to preferentially kill functionally defined leukemia stem cells, while sparing normal stem and progenitor cells. In addition to efficacy as single agents, flavaglines sensitize leukemia cells to several anticancer compounds, including front-line chemotherapeutic drugs used to treat leukemia patients. Mechanistic studies indicate that flavaglines strongly inhibit protein synthesis, leading to the reduction of short-lived antiapoptotic proteins. Notably though, treatment with flavaglines, alone or in combination with other drugs, yields a much stronger cytotoxic activity toward leukemia cells than the translational inhibitor temsirolimus. These results indicate that the underlying cell death mechanism of flavaglines is more complex than simply inhibiting general protein translation. Global gene expression profiling and cell biological assays identified Myc inhibition and the disruption of mitochondrial integrity to be features of flavaglines, which we propose contribute to their efficacy in targeting leukemia cells. Taken together, these findings indicate that rocaglamide and silvestrol are distinct from clinically available translational inhibitors and represent promising candidates for the treatment of leukemia.

AMPK/FIS1-Mediated Mitophagy Is Required for Self-Renewal of Human AML Stem Cells

Leukemia stem cells (LSCs) are thought to drive the genesis of acute myeloid leukemia (AML) as well as relapse following chemotherapy. Because of their unique biology, developing effective methods to eradicate LSCs has been a significant challenge. In the present study, we demonstrate that intrinsic overexpression of the mitochondrial dynamics regulator FIS1 mediates mitophagy activity that is essential for primitive AML cells. Depletion of FIS1 attenuates mitophagy and leads to inactivation of GSK3, myeloid differentiation, cell cycle arrest, and a profound loss of LSC self-renewal potential. Further, we report that the central metabolic stress regulator AMPK is also intrinsically activated in LSC populations and is upstream of FIS1. Inhibition of AMPK signaling recapitulates the biological effect of FIS1 loss. These data suggest a model in which LSCs co-opt AMPK/FIS1-mediated mitophagy as a means to maintain stem cell properties that may be otherwise compromised by the stresses induced by oncogenic transformation.

Targeted therapy for a subset of acute myeloid leukemias that lack expression of aldehyde dehydrogenase 1A1

Aldehyde dehydrogenase 1A1 (ALDH1A1) activity is high in hematopoietic stem cells and functions in part to protect stem cells from reactive aldehydes and other toxic compounds. In contrast, we found that approximately 25% of all acute myeloid leukemias expressed low or undetectable levels of ALDH1A1 and that this ALDH1A1− subset of leukemias correlates with good prognosis cytogenetics. ALDH1A1− cell lines as well as primary leukemia cells were found to be sensitive to treatment with compounds that directly and indirectly generate toxic ALDH substrates including 4-hydroxynonenal and the clinically relevant compounds arsenic trioxide and 4-hydroperoxycyclophosphamide. In contrast, normal hematopoietic stem cells were relatively resistant to these compounds. Using a murine xenotransplant model to emulate a clinical treatment strategy, established ALDH1A1− leukemias were also sensitive to in vivo treatment with cyclophosphamide combined with arsenic trioxide. These results demonstrate that targeting ALDH1A1− leukemic cells with toxic ALDH1A1 substrates such as arsenic and cyclophosphamide may be a novel targeted therapeutic strategy for this subset of acute myeloid leukemias.

Abstract 4041: IQGAP1 in human acutae myelogenous leukemia

Background: AML is phenotypically diverse. However, genome-wide sequencing studies indicate that median number of non-synonymous mutations in AML is 8 (B Vogelstein Science 2013) and that the same pathways are affected in tumors with distinct genetic alterations. These insights provided the impetus to confirm and extend the previously published observation that immunization with normal human white blood cells (WBC) whose surface charge had been modified in vitro by incubation with fluorodinitrobenzene (FDNB) elicited an antibody response that cross-reacted against a broad range of leukemias (Nature 232:197-198,1971). Specific Aims: 1) Isolation and molecular characterization of a shared antigenic moiety from human AML, 2) Examine the prevalence- and role in AML of IQGAP1, which was identified as a shared antigenic moiety. Methods: WBCs from healthy donors were incubated with FDNB at 10 4 molecules/cell for 12-15 minutes in PBS. Three rabbits were immunized with FDNB-treated cells (experimental rabbits). A control rabbit was immunized with sham-treated cells. After complement inactivation, immune sera were absorbed against WBCs from healthy donors. Absorbed immune sera were tested for their ability to stain AML cell lines by flow cytometry and clinical AML samples by Western blotting. Immunoprecipitation of antigens from whole cell lysates of clinical AML samples was done using IgG adsorbed on protein A/G Agarose beads. Liquid chromatography and mass spectrometry of the immuneprecipitated material was performed. Fold change in IQGAP1 expression in normal vs AML bone marrow was determined from raw data from Gene Expression Omnibus at the NCBI using Partek Genomic Suite. IQGAP1 expression was knocked down by shRNA and the effect on proliferation and colony formation was measured. Results: Sera from experimental rabbits stained AML cell lines with greater intensity by flow cytometry compared to serum from the control rabbit. Western blotting of whole cell lysates of clinical AML samples revealed bands that were recognized by immune serum from experimental rabbits but not the control rabbit. Immunoprecipitation of antigens from whole cell lysates of clinical AML samples revealed IQGAP1 as being differentially recognized in independent experiments. Western blots of human AML samples probed with anti-IQGAP1 antibody revealed the predicted 190 kDa band. The fold change in IQGAP1 expression in normal bone marrow versus AML was -3.22636, p-value 2.62 × 10e-7. Knocking down expression of IQGAP1 in K562, MV4-11 and THP1 cell lines resulted in significant decrease in proliferation and colony formation. Conclusion and Future Directions: IQGAP1 was identified as a shared antigenic moiety in. IQGAP1 is over-expressed in AML compared to normal bone marrow. Knocking down IQGAP1 expression in AML cell lines decreased proliferation and colony formation. Experiments to determine the mechanistic basis of the effect of FDNB on cells and if IQGAP1 is “druggable” are underway. Citation Format: Deepak M. Sahasrabudhe, Jeremy Bechelli, Fred P. Hagen, Mark Paris, Marlene Balys, Mohammad Minhajuddin, Jane Liesveld. IQGAP1 in human acutae myelogenous leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4041. doi:10.1158/1538-7445.AM2015-4041