Winnie F. Tam

STAT5 Is Crucial to Maintain Leukemic Stem Cells in Acute Myelogenous Leukemias Induced by MOZ-TIF2

MOZ-TIF2 is a leukemogenic fusion oncoprotein that confers self-renewal capability to hematopoietic progenitor cells and induces acute myelogenous leukemia (AML) with long latency in bone marrow transplantation assays. Here, we report that FLT3-ITD transforms hematopoietic cells in cooperation with MOZ-TIF2 in vitro and in vivo. Coexpression of FLT3-ITD confers growth factor independent survival/proliferation, shortens disease latency, and results in an increase in the number of leukemic stem cells (LSC). We show that STAT5, a major effector of aberrant FLT3-ITD signal transduction, is both necessary and sufficient for this cooperative effect. In addition, STAT5 signaling is essential for MOZ-TIF2-induced leukemic transformation itself. Lack of STAT5 in fetal liver cells caused rapid differentiation and loss of replating capacity of MOZ-TIF2-transduced cells enriched for LSCs. Furthermore, mice serially transplanted with Stat5(-/-) MOZ-TIF2 leukemic cells develop AML with longer disease latency and finally incomplete penetrance when compared with mice transplanted with Stat5(+/+) MOZ-TIF2 leukemic cells. These data suggest that STAT5AB is required for the self-renewal of LSCs and represents a combined signaling node of FLT3-ITD and MOZ-TIF2 driven leukemogenesis. Therefore, targeting aberrantly activated STAT5 or rewired downstream signaling pathways may be a promising therapeutic option.

Can FLT3 inhibitors overcome resistance in AML

The identification of FLT3 mutations across a range of the cytogenetic subgroups of AML has opened up the possibility of a targeted therapeutic approach with broad applicability. Four agents are currently in clinical trials, at least 3 of which have both sufficient activity against AML and sufficiently acceptable toxicity profiles to support continued efforts to refine their inclusion into therapeutic regimens for AML. Better understanding of the genetics of inherent and acquired resistance is needed to guide development of second-generation agents. Optimizing the integration of FLT3 inhibitor therapy with chemotherapy has the potential both to decrease toxicity and improve response.

Abstract 5509: Identification of synergistic drugs using combination high-throughput screening

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Increased understanding of the complexity of biological networks is forcing us to reconsider traditional views of disease and treatment. This is particularly true for cancer, as tumor cells have a tremendous capacity to evade the effects of drugs by mutation and/or pathway adaptation. We have developed a combination high throughput screening (cHTS) platform (robotics and analytics) that systematically and efficiently identifies combination drugs with synergistic activity. Synergistic combination drugs can increase maximal drug effect, increase potency and/or circumvent chemoresistance. In addition, coordinated action at multiple molecular targets can provide unique therapeutic benefit not achievable with the “one-drug, one-target” paradigm, especially problematic for cancer. We describe the application of cHTS to the discovery of novel synergistic drug combinations for the treatment of cancer. Using cHTS we have made the surprising discovery that A2A and β2 adrenergic receptor agonists strongly synergize with glucocorticoids (GCs) to inhibit the proliferation of hematologic malignancies. Using an 83 cell line panel, we find that synergistic activity is specific for select B-cell malignancies (including some cell lines that are GC-insensitive). These dexamethasone-dependent synergies are observed across a large panel of multiple myeloma (MM) cell lines and in multiple complex preclinical models of MM disease. In general, chemoresistance is a recurrent problem for cancer drugs and development of resistance after chronic exposure can reduce drug efficacy and promote refractory disease. We therefore examined the effects of chronic exposure to either A2A or β2AR agonists. Exposure of MM cells (MM.1S) to CGS-21680 (A2A agonist) or salmeterol (β2AR agonist) for one month reduced single agent sensitivity >80%. Surprisingly, combinations of either agent with dexamethasone maintained similar amounts of synergy and cell killing as found with naive untreated cells. Our studies demonstrate that synergistic combinations of A2A and β2AR agonists are highly selective for B-cell malignancies and support the notion that synergistic drug combinations can improve therapeutically relevant selectivity and circumvent drug resistance. These preclinical studies reinforce the rationale for investigation of A2A and β2AR agonists in the treatment of B-cell malignancies and in particular, patients who have MM. Furthermore, this work highlights the power of cHTS to interrogate combination activity across large panels of cancer cell lines with distinct molecular phenotypes and to identify novel mechanisms for therapeutic application. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5509.

Abstract 2215: Pharmacogenomic investigation of Bruton's tyrosine kinase (BTK) inhibitor ibrutinib (PCI-32765): drug sensitivity in diffuse large B-cell lymphoma (DLBCL) within a tumor microenvironment-aligned high-throughput screen.

Ibrutinib (PCI-32765) is an orally administered small molecule that covalently binds to Cys-481 of Bruton9s tyrosine kinase (BTK). Ibrutinib has demonstrated promise across several types of B-cell malignancies and is currently in Phase 2/3 clinical testing. Preclinical studies have established three key mechanisms following blunting of proximal B cell receptor (BCR) signaling through BTK inhibition: (1) suppression of pro-survival pathways, (2) diminished integrin activation and (3) attenuation of chemotactic response. Single agent ibrutinib Phase 1/2 clinical trials have revealed high clinical response rates in both naive and relapsed or refractory chronic lymphocytic leukemia (CLL), as well as, relapsed or refractory mantle cell lymphoma (MCL) patients (Byrd, et al. 2012 ASH; Wang, et al. 2012 ASH; Advani, et al. 2010 JCO). Response rates in a phase 2 diffuse large B cell lymphoma (DLBCL) clinical trial appeared to be more prevalent in “activated B-cell” (ABC) over “germinal center B-cell like” (GCB) DLBCL patients (Staudt et al., ASH 2012). To gain a further understanding of ibrutinib response and inform optimal therapeutic combinations in DLBCL, we established a combination high throughput pharmacology screen (cHTS) with ibrutinib. Ibrutinib was evaluated alone and in combination with 99 targeted compounds, across 17 (12 GCB; 5 ABC) DLBCL cell lines. To better align with human biology, DLBCL cell lines were screened in the presence of human marrow stromal cell conditioned media (hMSC-CM) and B-cell receptor stimulation via anti-IgG/anti-IgM antibodies. Interestingly, 8/17 (47%) DLBCL cell lines were intolerant to any external BCR stimulation, so those lines were screened in hMSC-CM without exogenous BCR stimulation. Under our experimental conditions, 11/17 (65%) cell lines displayed some sensitivity (IC 50 TM measurements [breakdown: 4/17 (24%) highly sensitive (IC 50 50 200 nM 50 1 M 50 > 10 M)]. Consistent with DLBCL clinical responses, the highly sensitive (IC 50 Citation Format: Cuc Davis, Tineke Casneuf, Willem Lightenberg, Richard Rickles, Winnie Tam, Matthias Versele, Steven McClue, Sriram Balasubramanian, Joseph Buggy, Kate Sasser, Brett Hall. Pharmacogenomic investigation of Bruton9s tyrosine kinase (BTK) inhibitor ibrutinib (PCI-32765): drug sensitivity in diffuse large B-cell lymphoma (DLBCL) within a tumor microenvironment-aligned high-throughput screen. [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 2215. doi:10.1158/1538-7445.AM2013-2215