Clayton A. Smith

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.

ALDHs in normal and malignant hematopoietic cells: Potential new avenues for treatment of AML and other blood cancers

Multiple studies have demonstrated that ALDH1A1 is elevated in hematopoietic stem cells (HSCs). As a means to better characterize such cells, we previously developed the fluorescent ALDH1A1 substrate Aldefluor to facilitate HSC identification and isolation. This has proven useful for counting and isolating HSCs from human bone marrow, peripheral blood and cord blood as well as stem cells in other tissues and organisms. Given the high level expression of ALDH1A1, we explored its biology and that of other ALDHs in HSCs and found that ALDH1A1 and ALDH3A1 were important in metabolizing reactive aldehydes (RAlds) and reactive oxygen species (ROS). In murine models, loss of these two isoforms resulted in a variety of effects on HSC biology, increased DNA damage and predisposition to leukemia formation when combined with a genetic driver of HSC proliferation and self-renewal. Loss of ALDH activity may also predispose to marrow failure and AML in Fanconis anemia (FA). ALDHs also have importance in mediating drug resistance in AML, may be useful in the identification of leukemia stem cells (LSCs) and ALDH activity levels may have prognostic significance. Together these findings suggest that further studying ALDH biology in AML and other blood cancers may provide important insights into malignant transformation and may point the way to the development of novel diagnostics and therapies.

Clonality of neutrophilia associated with plasma cell neoplasms: report of a SETBP1 mutation and analysis of a single institution series.

Abstract A rare but well-known association between plasma cell neoplasms and neutrophilia is known to exist. Whether the neutrophilia is secondary to the plasma cell neoplasm or this convergence represents two independent clonal disorders is unclear. We reviewed five consecutive cases from a single institution over a 3-year period, applying molecular, cytogenetic and cytokine-profiling approaches to determine whether neutrophilia associated with plasma cell neoplasms represents a reactive or clonal process. We report, for the first time, the occurrence of a SETBP1 mutation in two cases, as well as changes in G-CSF and IL-6 in SETBP1 wild type vs. mutated patients that are supportive of a hypothesis that neutrophilia associated with plasma cell neoplasms may sometimes be reactive and may sometimes represent a second clonal entity. Finally, using an ex vivo drug screening platform we report the potential efficacy of the multi-kinase inhibitor dasatinib in select patients.

Oncology scan--Autophagy and the radiation response.

Ionizing radiation is an effective therapy for the curafollowing inhibition of autophagy in the treated tissue. tive treatment or palliation of many forms of cancer; however, it is also associated with toxicity to normal tissues, both in the therapeutic setting and as a result of accidental exposure. Cellular responses to DNA damage produced by ionizing radiation include mitotic catastrophe, apoptosis, senescence, and autophagy. Although cell death following ionizing radiation is largely attributed to mitotic catastrophe and apoptosis, recent evidence has emerged regarding dual roles of autophagy as both a radiation-protective and radiationsensitizing response. Autophagy is a conserved mechanism for cellular homeostasis that is activated during times of stress to break down organelles, including damaged mitochondria and protein products, thereby providing additional energy sources for the cell. This is accomplished through production of a double-membrane autophagosome that fuses with lysosomes to form an autolysosome. It is well established that formation of the autophagosome is dependent on autophagy-related gene (ATG) products that assemble in a series of steps leading ultimately to the degradation of engulfed protein targets. These ATGs include BECLIN-1, ATG5, ATG7, and LC3-II among many others. BECLIN1 is normally bound by BCL-2, the anti-apoptotic protein, which prevents BECLIN-1 interaction with the phosphatidylinositol-3 kinase VPS34 for initiation of autophagosome formation. ATG5, ATG7, and LC3-II serve as crucial components for initiation and closure of the autophagosome. It has recently been proposed that autophagy can be divided into 4 functional categories including cytoprotective, cytotoxic, cytostatic, and nonprotective (1). These functional differences are based largely on the response produced within cells following a stressor, and this is often experimentally demonstrated by observing the effects of pharmacologic or genetic inhibition of autophagy on cellular response. A cytoprotective function for autophagy is observed when sensitivity to the stressor (eg, chemotherapeutic agent or irradiation) increases

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.

Preclinical Advances in Combined-Modality Cancer Immunotherapy With Radiation Therapy

Cancer immunotherapy as a field seeks to exploit the pathways by which tumors and their microenvironment escape detection and elimination by the host immune system. Cancer cells have demonstrated a variety of ways of escaping immune detection, including decreased presentation of tumor-specific antigens, release of immunosuppressive cytokines, and activation of T-cell inhibitory pathways. Interventions against the latter mechanism have demonstrated the greatest potential of cancer immunotherapy to date. The cytotoxic T-lymphocyte antigen 4 (CTLA4) and programmed cell death 1 receptor (PD-1) are T-cell surface receptors that, when activated by their respective ligands, serve to suppress T-cell functions by separate pathways. Anti-CTLA4 (ipilimumab) and anti-PD1 (nivolumab) have been developed as immune checkpoint inhibitors and approved by the US Food and Drug Administration for clinical use in the treatment of advanced melanoma (ipilimumab and nivolumab) and metastatic squamous non-small cell lung cancer (nivolumab). The finding that dual immune checkpoint blockade in patients with metastatic melanoma prolongs overall survival and induces greater responses over monotherapy advances the promise of immunotherapy as a treatment modality (1). Radiation therapy has long been used as a local treatment modality in cancer care. The cytotoxic effect of radiation results in release of tumor-associated antigens and damage-associated molecular patterns that can produce an immunogenic response, which in some cases can produce an abscopal effect. The earliest report of an abscopal effect, defined as an “effect of radiation that is manifested at a distance from the volume of tissue which is directly irradiated,” listed in PubMed.gov was authored by Law and Mole in 1961 (2). The earliest evidence that we are aware of that abscopal effects involved immune responses was by Mikuriya et al (3), who found that large doses of ionizing radiation could be leveraged into a systemic antitumor response. The initial report by Postow

Targeting Enox1 in tumor stroma increases the efficacy of fractionated radiotherapy

The goal of this investigation was to clarify the question of whether targeting Enox1 in tumor stroma would synergistically enhance the survival of tumor-bearing mice treated with fractionated radiotherapy. Enox1, a NADH oxidase, is expressed in tumor vasculature and stroma. However, it is not expressed in many tumor types, including HT-29 colorectal carcinoma cells. Pharmacological inhibition of Enox1 in endothelial cells inhibited repair of DNA double strand breaks, as measured by γH2AX and 53BP1 foci formation, as well as neutral comet assays. For 4 consecutive days athymic mice bearing HT-29 hindlimb xenografts were injected with a small molecule inhibitor of Enox1 or solvent control. Tumors were then administered 2 Gy of x-rays. On day 5 tumors were administered a single ‘top-up’ fraction of 30 Gy, the purpose of which was to amplify intrinsic differences in the radiation fractionation regimen produced by Enox1 targeting. Pharmacological targeting of Enox1 resulted in 80% of the tumor-bearing mice surviving at 90 days compared to only 40% of tumor-bearing mice treated with solvent control. The increase in survival was not a consequence of reoxygenation, as measured by pimonidazole immunostaining. These results are interpreted to indicate that targeting of Enox1 in tumor stroma significantly enhances the effectiveness of 2 Gy fractionated radiotherapy and identifies Enox1 as a potential therapeutic target.