Alexandra N. Christodoulou

The Public Repository of Xenografts Enables Discovery and Randomized Phase II-like Trials in Mice

More than 90% of drugs with preclinical activity fail in human trials, largely due to insufficient efficacy. We hypothesized that adequately powered trials of patient-derived xenografts (PDX) in mice could efficiently define therapeutic activity across heterogeneous tumors. To address this hypothesis, we established a large, publicly available repository of well-characterized leukemia and lymphoma PDXs that undergo orthotopic engraftment, called the Public Repository of Xenografts (PRoXe). PRoXe includes all de-identified information relevant to the primary specimens and the PDXs derived from them. Using this repository, we demonstrate that large studies of acute leukemia PDXs that mimic human randomized clinical trials can characterize drug efficacy and generate transcriptional, functional, and proteomic biomarkers in both treatment-naive and relapsed/refractory disease.

Co-activation of AMPK and mTORC1 Induces Cytotoxicity in Acute Myeloid Leukemia

AMPK is a master regulator of cellular metabolism that exerts either oncogenic or tumor suppressor activity depending on context. Here, we report that the specific AMPK agonist GSK621 selectively kills acute myeloid leukemia (AML) cells but spares normal hematopoietic progenitors. This differential sensitivity results from a unique synthetic lethal interaction involving concurrent activation of AMPK and mTORC1. Strikingly, the lethality of GSK621 in primary AML cells and AML cell lines is abrogated by chemical or genetic ablation of mTORC1 signaling. The same synthetic lethality between AMPK and mTORC1 activation is established in CD34-positive hematopoietic progenitors by constitutive activation of AKT or enhanced in AML cells by deletion of TSC2. Finally, cytotoxicity in AML cells from GSK621 involves the eIF2α/ATF4 signaling pathway that specifically results from mTORC1 activation. AMPK activation may represent a therapeutic opportunity in mTORC1-overactivated cancers.

Characterization of activating mutations of NOTCH3 in T-cell acute lymphoblastic leukemia and anti-leukemic activity of NOTCH3 inhibitory antibodies

Notch receptors have been implicated as oncogenic drivers in several cancers, the most notable example being NOTCH1 in T-cell acute lymphoblastic leukemia (T-ALL). To characterize the role of activated NOTCH3 in cancer, we generated an antibody that detects the neo-epitope created upon gamma-secretase cleavage of NOTCH3 to release its intracellular domain (ICD3), and sequenced the negative regulatory region (NRR) and PEST (proline, glutamate, serine, threonine) domain coding regions of NOTCH3 in a panel of cell lines. We also characterize NOTCH3 tumor-associated mutations that result in activation of signaling and report new inhibitory antibodies. We determined the structural basis for receptor inhibition by obtaining the first co-crystal structure of a NOTCH3 antibody with the NRR protein and defined two distinct epitopes for NRR antibodies. The antibodies exhibit potent anti-leukemic activity in cell lines and tumor xenografts harboring NOTCH3 activating mutations. Screening of primary T-ALL samples reveals that 2 of 40 tumors examined show active NOTCH3 signaling. We also identified evidence of NOTCH3 activation in 12 of 24 patient-derived orthotopic xenograft models, 2 of which exhibit activation of NOTCH3 without activation of NOTCH1. Our studies provide additional insights into NOTCH3 activation and offer a path forward for identification of cancers that are likely to respond to therapy with NOTCH3 selective inhibitory antibodies.

Erratum: The Public Repository of Xenografts Enables Discovery and Randomized Phase II-like Trials in Mice (Cancer Cell (2016) 29 (574–586))

Elizabeth C. Townsend, Mark A. Murakami, Alexandra Christodoulou, Amanda L. Christie, Johannes Köster, Tiffany A. DeSouza, Elizabeth A. Morgan, Scott P. Kallgren, Huiyun Liu, Shuo-Chieh Wu, Olivia Plana, Joan Montero, Kristen E. Stevenson, Prakash Rao, Raga Vadhi, Michael Andreeff, Philippe Armand, Karen K. Ballen, Patrizia Barzaghi-Rinaudo, Sarah Cahill, Rachael A. Clark, Vesselina G. Cooke, Matthew S. Davids, Daniel J. DeAngelo, David M. Dorfman, Hilary Eaton, Benjamin L. Ebert, Julia Etchin, Brant Firestone, David C. Fisher, Arnold S. Freedman, Ilene A. Galinsky, Hui Gao, Jacqueline S. Garcia, Francine Garnache-Ottou, Timothy A. Graubert, Alejandro Gutierrez, Ensar Halilovic, Marian H. Harris, Zachary T. Herbert, Steven M. Horwitz, Giorgio Inghirami, Andrew M. Intlekofer, Moriko Ito, Shai Izraeli, Eric D. Jacobsen, Caron A. Jacobson, Sébastien Jeay, Irmela Jeremias, Michelle A. Kelliher, Raphael Koch, Marina Konopleva, Nadja Kopp, Steven M. Kornblau, Andrew L. Kung, Thomas S. Kupper, Nicole R. LeBoeuf, Ann S. LaCasce, Emma Lees, Loretta S. Li, A. Thomas Look, Masato Murakami, Markus Muschen, Donna Neuberg, Samuel Y. Ng, Oreofe O. Odejide, Stuart H. Orkin, Rachel R. Paquette, Andrew E. Place, Justine E. Roderick, Jeremy A. Ryan, Stephen E. Sallan, Brent Shoji, Lewis B. Silverman, Robert J. Soiffer, David P. Steensma, Kimberly Stegmaier, Richard M. Stone, Jerome Tamburini, Aaron R. Thorner, Paul van Hummelen, Martha Wadleigh, Marion Wiesmann, Andrew P. Weng, Jens U. Wuerthner, David A. Williams, Bruce M. Wollison, Andrew A. Lane, Anthony Letai, Monica M. Bertagnolli, Jerome Ritz, Myles Brown, Henry Long, Jon C. Aster, Margaret A. Shipp, James D. Griffin, and David M. Weinstock* *Correspondence: dweinstock@partners.org http://dx.doi.org/10.1016/j.ccell.2016.06.008

Inhibition of USP10 induces degradation of oncogenic FLT3

Oncogenic forms of the kinase FLT3 are important therapeutic targets in acute myeloid leukemia (AML); however, clinical responses to small-molecule kinase inhibitors are short-lived as a result of the rapid emergence of resistance due to point mutations or compensatory increases in FLT3 expression. We sought to develop a complementary pharmacological approach whereby proteasome-mediated FLT3 degradation could be promoted by inhibitors of the deubiquitinating enzymes (DUBs) responsible for cleaving ubiquitin from FLT3. Because the relevant DUBs for FLT3 are not known, we assembled a focused library of most reported small-molecule DUB inhibitors and carried out a cellular phenotypic screen to identify compounds that could induce the degradation of oncogenic FLT3. Subsequent target deconvolution efforts allowed us to identify USP10 as the critical DUB required to stabilize FLT3. Targeting of USP10 showed efficacy in preclinical models of mutant-FLT3 AML, including cell lines, primary patient specimens and mouse models of oncogenic-FLT3-driven leukemia.

Discovery of a highly potent FLT3 kinase inhibitor for FLT3-ITD-positive AML.

TH was responsible for the cytomorphologic analysis and was the principle investigator of the study. AS contributed to the cytogenetics, SE to the cytomorphology, KP to the mutation analyses and TA to the collection of clinical data. WK was involved in the statistical analyses. CH was responsible for the cytogenetics. MM investigated the molecular mutations, analyzed the data and wrote the manuscript. All authors read and contributed to the final version of the manuscript.

Abstract A23: Type II JAK2 inhibitor NVP-CHZ868 has potent activity in JAK2-dependent B-cell acute lymphoblastic leukemias (B-ALLs) in vivo.

Approximately 10% of B-ALLs harbor CRLF2 rearrangements and have a poor prognosis. Although these leukemias are addicted to JAK2 signaling, ATP-competitive type I JAK2 inhibitors have limited activity (Weigert et al . J Exp Med 2012). This may result from heterodimerization of JAK2 with other JAK family members (Koppikar et al . Nature 2012). Type II inhibitors bind JAK2 in the inactive conformation and may have non-cross resistance with type I inhibitors. In Ba/F3 cells dependent on CRLF2 and the gain-of-function allele JAK2 R683G, the type II JAK2 inhibitor NVP-CHZ868 was more potent (IC50 21nM) than the type I inhibitors NVP-BSK805 (IC50 443nM) and NVP-BVB808 (IC50 111nM). Unlike type I inhibitors, CHZ868 completely abrogated JAK2 and STAT5 phosphorylation. In addition, the JAK2 Y931C allele that confers 4-6-fold resistance to BSK805 and BVB808 did not affect the IC50 of CHZ868. We assessed in vivo efficacy of CHZ868 in mice transplanted with transgenic (CRLF2/JAK2 R683G/Cdkn2a -/- or CRLF2/JAK2 R683G/Pax5 +/- /Ts1Rhr) or primary human CRLF2-rearranged B-ALLs. Mice treated for 5-6 days with CHZ868 (30mg/kg/day PO) had significant reductions in spleen size compared to control mice and complete loss of phospho-STAT5 in residual leukemia cells. In both murine leukemias and human xenografts, CHZ868 prolonged survival compared to controls (p 30) clones sequenced harbored the same JAK2 L884P mutation. Ba/F3 cells expressing CRLF2 with JAK2 R683G/L884P had 14-fold resistance to CHZ868 (R683G IC50 16nM; R683G/L884P IC50 231nM). JAK2 L884P is homologous to an EGFR L747P activating mutation (He et al . Clin Cancer Res 2012), which destabilizes the P-loop and C-helix portion of the kinase domain. Next-generation sequencing of JAK2 from splenocytes of mice that progressed on CHZ868 treatment did not identify L884P or other missense mutations at >1% frequency, suggesting in vivo treatment failure was not due to JAK2 mutation. To improve CHZ868 efficacy, we tested for synergy with multiple chemotherapy agents in MHH-CALL4 cells, which harbor a CRLF2/IGH rearrangement and JAK2 I682F mutation. Among the tested agents, dexamethasone was highly synergistic with CHZ868. In mice transplanted with CRLF2/JAK2 R683G/Pax5 +/- /Ts1Rhr murine B-ALL, a 14-day course of CHZ868 prolonged survival compared to vehicle (p in vitro and in vivo . Thus, combination strategies using dexamethasone with type II JAK2 inhibitors merit testing in patients with relapsed/refractory, JAK2-dependent B-ALL. Citation Format: Loretta S. Li, Nadja Kopp, Shuo-Chieh Wu, Jordy Van Der Zwet, Jacob V. Layer, Oliver Weigert, Amanda L. Christie, Alexandra N. Christodoulou, Huiyun Liu, Akinori Yoda, Thomas Radimerski, David M. Weinstock. Type II JAK2 inhibitor NVP-CHZ868 has potent activity in JAK2-dependent B-cell acute lymphoblastic leukemias (B-ALLs) in vivo. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr A23.