Brett M. Stevens

Inhibition of redox/Fyn/c-Cbl pathway function by Cdc42 controls tumour initiation capacity and tamoxifen sensitivity in basal-like breast cancer cells

We found that basal‐like breast cancer (BLBC) cells use Cdc42 to inhibit function of the redox/Fyn/c‐Cbl (RFC) pathway, which normally functions to convert small increases in oxidative status into enhanced degradation of c‐Cbl target proteins. Restoration of RFC pathway function by genetic or pharmacological Cdc42 inhibition enabled harnessing of pro‐oxidant effects of low µM tamoxifen (TMX) concentrations – concentrations utilized in trials on multiple tumour types – to suppress division and induce death of BLBC cells in vitro and to confer TMX sensitivity in vivo through oestrogen receptor‐α‐independent mechanisms. Cdc42 knockdown also inhibited generation of mammospheres in vitro and tumours in vivo, demonstrating the additional importance of this pathway in tumour initiating cell (TIC) function. These findings provide a new regulatory pathway that is subverted in cancer cells, a novel means of attacking TIC and non‐TIC aspects of BLBCs, a lead molecule (ML141) that confers sensitivity to low µM TMX in vitro and in vivo and also appear to be novel in enhancing sensitivity to a non‐canonical mode of action of an established therapeutic agent.

The biologic properties of leukemias arising from BCR/ABL-mediated transformation vary as a function of developmental origin and activity of the p19ARF gene

Recent reports have shown that upon expression of appropriate oncogenes, both stem cells and more differentiated progenitor populations can serve as leukemia-initiating cells. These studies suggest that oncogenic mutations subvert normal development and induce reacquisition of stem-like features. However, no study has described how specific mutations influence the ability of differentiating cell subsets to serve as leukemia-initiating cells and if varying such cellular origins confers a functional difference. We have examined the role of the tumor suppressor gene p19(ARF) in a murine model of acute lymphoblastic leukemia and found that loss of p19(ARF) changes the spectrum of cells capable of tumor initiation. With intact p19(ARF), only hematopoietic stem cells (HSCs) can be directly transformed by BCR/ABL expression. In a p19(ARF)-null genetic background expression of the BCR/ABL fusion protein renders functionally defined HSCs, common lymphoid progenitors (CLP), and precursor B-lymphocytes competent to generate leukemia stem cells. Furthermore, we show that leukemias arising from p19(ARF)-null HSC versus pro-B cells differ biologically, including relative response to drug insult. Our observations elucidate a unique mechanism by which heterogeneity arises in tumor populations harboring identical genetic lesions and show that activity of p19(ARF) profoundly influences the nature of tumor-initiating cells during BCR/ABL-mediated leukemogenesis.

MLL1 and DOT1L cooperate with meningioma-1 to induce acute myeloid leukemia

Meningioma-1 (MN1) overexpression is frequently observed in patients with acute myeloid leukemia (AML) and is predictive of poor prognosis. In murine models, forced expression of MN1 in hematopoietic progenitors induces an aggressive myeloid leukemia that is strictly dependent on a defined gene expression program in the cell of origin, which includes the homeobox genes Hoxa9 and Meis1 as key components. Here, we have shown that this program is controlled by two histone methyltransferases, MLL1 and DOT1L, as deletion of either Mll1 or Dot1l in MN1-expressing cells abrogated the cell of origin-derived gene expression program, including the expression of Hoxa cluster genes. In murine models, genetic inactivation of either Mll1 or Dot1l impaired MN1-mediated leukemogenesis. We determined that HOXA9 and MEIS1 are coexpressed with MN1 in a subset of clinical MN1hi leukemia, and human MN1hi/HOXA9hi leukemias were sensitive to pharmacologic inhibition of DOT1L. Together, these data point to DOT1L as a potential therapeutic target in MN1hi AML. In addition, our findings suggest that epigenetic modulation of the interplay between an oncogenic lesion and its cooperating developmental program has therapeutic potential in AML.

Redox biology in normal cells and cancer: restoring function of the redox/Fyn/c-Cbl pathway in cancer cells offers new approaches to cancer treatment.

This review discusses a unique discovery path starting with novel findings on redox regulation of precursor cell and signaling pathway function and identification of a new mechanism by which relatively small changes in redox status can control entire signaling networks that regulate self-renewal, differentiation, and survival. The pathway central to this work, the redox/Fyn/c-Cbl (RFC) pathway, converts small increases in oxidative status to pan-activation of the c-Cbl ubiquitin ligase, which controls multiple receptors and other proteins of central importance in precursor cell and cancer cell function. Integration of work on the RFC pathway with attempts to understand how treatment with systemic chemotherapy causes neurological problems led to the discovery that glioblastomas (GBMs) and basal-like breast cancers (BLBCs) inhibit c-Cbl function through altered utilization of the cytoskeletal regulators Cool-1/βpix and Cdc42, respectively. Inhibition of these proteins to restore normal c-Cbl function suppresses cancer cell division, increases sensitivity to chemotherapy, disrupts tumor-initiating cell (TIC) activity in GBMs and BLBCs, controls multiple critical TIC regulators, and also allows targeting of non-TICs. Moreover, these manipulations do not increase chemosensitivity or suppress division of nontransformed cells. Restoration of normal c-Cbl function also allows more effective harnessing of estrogen receptor-α (ERα)-independent activities of tamoxifen to activate the RFC pathway and target ERα-negative cancer cells. Our work thus provides a discovery strategy that reveals mechanisms and therapeutic targets that cannot be deduced by standard genetics analyses, which fail to reveal the metabolic information, isoform shifts, protein activation, protein complexes, and protein degradation critical to our discoveries.

Cool‐1‐Mediated Inhibition of c‐Cbl Modulates Multiple Critical Properties of Glioblastomas, Including the Ability to Generate Tumors In Vivo

We discovered that glioblastoma (GBM) cells use Cool‐1/β‐pix to inhibit normal activation of the c‐Cbl ubiquitin ligase via the redox/Fyn/c‐Cbl pathway and that c‐Cbl inhibition is critical for GBM cell function. Restoring normal c‐Cbl activity by Cool‐1 knockdown in vitro reduced GBM cell division, almost eliminated generation of adhesion‐independent spheroids, reduced the representation of cells expressing antigens thought to identify tumor initiating cells (TICs), reduced levels of several proteins of critical importance in TIC function (such as Notch‐1 and Sox2), and increased sensitivity to BCNU (carmustine) and temozolomide (TMZ). In vivo, Cool‐1 knockdown greatly suppressed the ability of GBM cells to generate tumors, an outcome that was c‐Cbl dependent. In contrast, Cool‐1 knockdown did not reduce division or increase BCNU or TMZ sensitivity in primary glial progenitor cells and Cool‐1/c‐Cbl complexes were not found in normal brain tissue. Our studies provide the first evidence that Cool‐1 may be critical in the biology of human tumors, that suppression of c‐Cbl by Cool‐1 may be critical for generation of at least a subset of GBMs and offer a novel target that appears to be selectively necessary for TIC function and modulates chemoresistance in GBM cells. Targeting such proteins that inhibit c‐Cbl offers potentially attractive opportunities for therapeutic development. Stem Cells 2014;32:1124–1135

Characterization and targeting of malignant stem cells in patients with advanced myelodysplastic syndromes

Myelodysplastic syndrome (MDS) is a chronic hematologic disorder that frequently evolves to more aggressive stages and in some cases leads to acute myeloid leukemia (AML). MDS arises from mutations in hematopoietic stem cells (HSCs). Thus, to define optimal therapies, it is essential to understand molecular events driving HSC pathogenesis. In this study, we report that during evolution of MDS, malignant HSCs activate distinct cellular programs that render such cells susceptible to therapeutic intervention. Specifically, metabolic analyses of the MDS stem cell compartment show a profound activation of protein synthesis machinery and increased oxidative phosphorylation. Pharmacological targeting of protein synthesis and oxidative phosphorylation demonstrated potent and selective eradication of MDS stem cells in primary human patient specimens. Taken together, our findings indicate that MDS stem cells are reliant on specific metabolic events and that such properties can be targeted prior to the onset of clinically significant AML, during antecedent MDS.Myelodysplastic syndrome (MDS) arises from mutations in hematopoietic stem cells (HSCs). Here, the authors demonstrate that HSCs in higher-risk MDS express the surface marker CD123 and are characterized by activation of protein synthesis machinery and increased oxidative phosphorylation.

Abstract B14: Targeting a network of cancer control nodes through rescue of c-Cbl: A novel therapeutic approach in GBM

Glioblastoma multiforme is the most common primary brain tumor in adults, the most malignant of all intracranial tumors, and is associated with inevitable recurrence and 15 month mean survival despite multimodal therapy. The majority of GBM tumors maintain heterogeneous amplifications (many in the absence of genetic mutation) in receptor tyrosine kinases (RTKs), which are mediators of growth factor signaling. Our lab has discovered that control over RTK levels in normal CNS progenitors is maintained by the Redox/Fyn/c-Cbl (RFC) pathway whereby increased oxidative state results in activation of the E3 ubiquitin ligase c-Cbl and the subsequent internalization and degradation of target RTKs. RFC signaling is disrupted in GBM due to a physical sequestration of c-Cbl, preventing pro-oxidative chemotherapeutics from reducing pro-survival signaling through Epidermal Growth Factor Receptor, Platelet-Derived Growth Factor Receptor, etc. Using a triangulating drug screen, we identified candidate FDA-approved small molecules capable of reducing signaling downstream of c-Cbl-targeted RTKs. We found that candidate URMC-06, an antidepressant with a good clinical safety profile, could rescue RFC signaling and significantly enhance the sensitivity of GBM cells to a pro-oxidative agent (Tamoxifen) in a c-Cbl-dependent manner. There is significant evidence to suggest that a tumor initiating cell population mediates the malignancy of GBM tumors. These cells express stem-cell associated markers and exhibit self-renewal capacity. We observed a c-Cbl-dependent inhibition of neurosphere formation (an in vitro measure of self-renewal) and a reduction in the expression of stem cell markers Sox2, beta-catenin, FoxM1 and Nestin in GBM cells treated with URMC-06. Our candidate also facilitated reduction in heat shock signaling and an inhibition of autophagy (both putative survival mechanisms) in GBM cells. URMC-06 was effective, in combination with clinically relevant agents (Temozolomide and Tamoxifen), at reducing tumor progression in an in vivo xenograft model of GBM. Our work supports the hypothesis that c-Cbl is a critical player in GBM that integrates multiple nodes of cancer control and that pharmacological rescue of c-Cbl represents a potentially powerful therapeutic approach. Citation Format: Jennifer L. Stripay, Brett M. Stevens, Addie L. Bardin, Mark D. Noble. Targeting a network of cancer control nodes through rescue of c-Cbl: A novel therapeutic approach in GBM. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr B14.

Abstract 2912: Inhibition of redox/Fyn/c-Cbl pathway function by Cdc42 controls tumor initiation capacity and tamoxifen sensitivity in basal-like breast cancer cells.

Two of the substantial challenges in cancer research are to identify means overcoming resistance of malignant cells to existing therapies and to eliminate the unique small population within bulk tumors, named tumor initiating cells or cancer stem cells which have been proposed to regenerate resistant relapsed tumors. In this study, we offer a novel therapeutic target relevant to both of these goals on basal-like (triple-negative) breast cancer (BLBC) cells, one of the most challenging cancers to treat with current regimens. Our findings emerged from examining the puzzling differences in regulation of activation of the E3 ubiqutin ligase c-Cbl in normal cells and in cancer cells by low microM concentrations of tamoxifen (TMX). In normal progenitor cells, TMX causes oxidation and sequential activation of Fyn kinase and the c-Cbl ubiquitin ligase. Activation of c-Cbl via this redox/Fyn/c-Cbl (RFC) pathway leads to accelerated degradation of receptor tyrosine kinases that are critical in cell survival and division (such as the epidermal growth factor (EGFR)). In BLBC cells, in contrast, TMX does not activate c-Cbl despite making the cells more oxidized and causing activation of Fyn. Moreover, BLBC cells exposed to TMX show no reduction in levels of EGFR. We found that TMX-induced activation of c-Cbl in BLBC cells was inhibited due to expression of Cdc42. Cdc42 sequestered c-Cbl, prevented its activation and prevented EGFR from being degraded. Restoration of c-Cbl function, by inhibiting activation of Cdc42, reduced EGFR levels in these cells. More critically, restoring c-Cbl function sensitized these cells to TMX both in vitro and in vivo through estrogen receptor-independent mechanisms. Analysis of tumor growth and formation in vivo showed that reducing the levels of Cdc42 reduced the size of tumors, increased sensitivity to TMX and decreased tumor forming capacity of these cells. The results provide a novel defense mechanism that BLBC cells utilize to prevent EGFR degradation, which may have high relevance to treatment of these tumors. Of particular importance is the ability of cdc42 to confer TMX sensitivity on these otherwise resistant tumor cells. Moreover, as use of low microM levels of TMX has been attempted for over a dozen different kinds of cancers, at least some of which express increased levels of Cdc42, this strategy may be relevant to the treatment of multiple different cancers. Citation Format: Hsing-Yu Chen, Yin Yang, Brett Stevens, Mark Noble. Inhibition of redox/Fyn/c-Cbl pathway function by Cdc42 controls tumor initiation capacity and tamoxifen sensitivity in basal-like breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2912. doi:10.1158/1538-7445.AM2013-2912

Abstract 4106: Cool-1-mediated inhibition of c-Cbl as a therapuetic target, which modulates multiple critical properties of glioblastomas.

Glioblastomas (GBMs), the most malignant glial tumors, differ from normal glial progenitor cells in many ways, but little is known about the molecular circuitry underlying these differences. We have discovered a novel means by which GBMs become chemo-resistant, based on inhibition of the redox/Fyn/c-Cbl pathway by overexpression of Cool-1(Beta-Pix). The protein c-Cbl, an E3 ubiquitin ligase, is responsible for the ubiquitination and degradation of multiple receptor tyrosine kinases critical for cell division and cell survival. In normal glial progenitor cells of the CNS, exposure to chemotherapy oxidizes cells, leading to sequential activation of Fyn and c-Cbl. However in GBM cells, exposure to BCNU or other chemical pro-oxidants does not lead to c-Cbl activation. Further studies demonstrated that these agents do cause Fyn activation, suggesting that GBMs inhibit c-Cbl phosphorylation leading to decreased degradation of EGFR, accounting for increased EGFR signaling. We next discovered that the decrease in c-Cbl activation via the redox/Fyn/c-Cbl pathway was due to c-Cbl sequestration by Cool-1, which is overexpressed in GBM cells and samples of tumors. We found that oxidant-associated c-Cbl activation can be restored by shRNA-mediated inhibition of Cool-1 expression, leading to restoration of normal c-Cbl-mediated degradation of RTKs (including EGFR). We further found that restoration of Cool-1 activity modulated multiple other hallmarks of GBM, including changes in migration, cell cycle parameters, and altering the cancer stem cell phenotype of our cells. In contrast, Cool-1/c-Cbl complexes were not found in normal brain or normal glial progenitors and Cool-1 knockdown did not reduce progenitor cell division. The translation of these findings into an in vivo intracranial xenograft model of GBM showed that Cool-1 is essential for tumorigenesis and decreasing Cool-1 levels leads to decreased tumor take, decreased tumor growth, and increased survival. In sum, the ability of Cool-1/c-Cbl interactions to modulate a variety of tumor cell properties suggests that these interactions offer an attractive target for modulating GBM growth, particularly due to the absence of Cool-1/c-Cbl complexes in normal brain tissue and to the lack of effects of Cool-1 knockdown on division of normal glial progenitor cells. The activation of c-Cbl activity through pharmacological agents is a potentially viable strategy for the specific eradication of GBM cells. Citation Format: Brett M. Stevens, Christopher J. Folts, Warner Chen, Addie L. Bardin, Mark Noble. Cool-1-mediated inhibition of c-Cbl as a therapuetic target, which modulates multiple critical properties of glioblastomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4106. doi:10.1158/1538-7445.AM2013-4106

Abstract 2074: Cool-1-mediated inhibition of c-Cbl modulates multiple critical properties of glioblastomas, including the ability to generate tumors in vivo

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Glioblastomas (GBMs), the most malignant glial tumors, differ from normal glial progenitor cells in many ways, but little is known about the molecular circuitry underlying these differences. We have discovered a novel means by which GBMs become chemo-resistant, based on inhibition of the redox/Fyn/c-Cbl pathway by overexpression of Cool-1. The protein c-Cbl, an E3 ubiquitin ligase, is responsible for the ubiquitination and degradation of multiple receptor tyrosine kinases critical for cell division and cell survival. In normal glial progenitor cells of the CNS, exposure to chemotherapy oxidizes cells, leading to sequential activation of Fyn and c-Cbl. However in GBM cells, exposure to BCNU or other chemical pro-oxidants does not lead to c-Cbl activation. Further studies have demonstrated that these agents do cause Fyn activation, suggesting that GBMs inhibit c-Cbl phosphorylation leading to decreased degradation of EGFR, accounting for increased EGFR signaling. We therefore set out to investigate the factors involved in the decreased c-Cbl activity specific to GBMs. In this work we discovered that oxidant-associated c-Cbl activation can be restored by suppressing expression of the protein Cool-1, found to be over-expressed in GBM. Our data further demonstrate that Cool-1 and c-Cbl formed complexes, that genetic reduction in Cool-1 levels restored normal c-Cbl-mediated degradation of RTKs (including EGFR) in response to exposure to pro-oxidants, and that restoration of normal c-Cbl activation has multiple effects on hallmarks of GBM. Cool-1 decreases lead to decreased migration and decreased numbers of cells in the cell cycle. Cool-1 inhibition leads to decreased number of cells that exhibit cancer stem cells characteristics including decreased antigen positivity and decreased sphere forming ability. In contrast, Cool-1/c-Cbl complexes were not found in normal brain or normal glial progenitors and Cool-1 knockdown did not reduce progenitor division. The translation of these findings into an in vivo intracranial xenograft model of GBM shows that Cool-1 is essential for tumorigenesis and decreasing Cool-1 levels leads to decreased tumor take, decreased tumor growth, and increased survival. In sum, the ability of Cool-1/c-Cbl interactions, to modulate a variety of tumor cell properties suggests that these interactions offer an attractive target for modulating GBM growth, particularly due to the absence of Cool-1/c-Cbl complexes in normal brain tissue and to the lack of effects of Cool-1 knockdown on division of normal glial progenitor cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2074. doi:1538-7445.AM2012-2074