Loretta S. Li

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.

The intersection of genetic and chemical genomic screens identifies GSK-3α as a target in human acute myeloid leukemia

Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults. Long-term survival of patients with AML has changed little over the past decade, necessitating the identification and validation of new AML targets. Integration of genomic approaches with small-molecule and genetically based high-throughput screening holds the promise of improved discovery of candidate targets for cancer therapy. Here, we identified a role for glycogen synthase kinase 3α (GSK-3α) in AML by performing 2 independent small-molecule library screens and an shRNA screen for perturbations that induced a differentiation expression signature in AML cells. GSK-3 is a serine-threonine kinase involved in diverse cellular processes, including differentiation, signal transduction, cell cycle regulation, and proliferation. We demonstrated that specific loss of GSK-3α induced differentiation in AML by multiple measurements, including induction of gene expression signatures, morphological changes, and cell surface markers consistent with myeloid maturation. GSK-3α-specific suppression also led to impaired growth and proliferation in vitro, induction of apoptosis, loss of colony formation in methylcellulose, and anti-AML activity in vivo. Although the role of GSK-3β has been well studied in cancer development, these studies support a role for GSK-3α in AML.

Characterization of genomic breakpoints in MLL and CBP in leukemia patients with t(11;16)

The recurring chromosome translocation t(11;16)(q23;p13) is detected in leukemia patients, virtually all of whom have received previous chemotherapy with topoisomerase (topo) II inhibitors. In the t(11;16), 3′ CBP, on 16p13, is fused to 5′ MLL, on 11q23, resulting in an MLL–CBP fusion gene that plays an important role in leukemogenesis. In this study, we cloned genomic breakpoints of the MLL and CBP genes in the t(11;16) in the SN‐1 cell line and in five patients with therapy‐related leukemia, all of whom had received topo II inhibitors for previous tumors. In all patients except one, both the genomic MLL–CBP and the reciprocal fusions were cloned. Genomic breakpoints in MLL occurred in the 8.3‐kb breakpoint cluster region in all patients, whereas the breakpoints in CBP clustered in an 8.2‐kb region of intron 3 in four patients. Genomic breakpoints in MLL occurred in intron 11 near the topo II cleavage site in the SN‐1 cell line and in one patient, and they were close to LINE repetitive sequences in two other patients. In the remaining two patients, genomic breakpoints were in intron 9 in Alu repeats. Genomic breakpoints in CBP occurred in and around Alu repeats in one and two patients, respectively. In two patients, the breaks were near LINE repetitive sequences, suggesting that repetitive DNA sequences may play a role. No specific recombination motifs were identified at or near the breakpoint junctions. No topo II cleavage sites were detected in introns 2 and 3 of CBP. However, there were deletions and duplications at the breakpoints in both MLL and CBP and microhomologies or nontemplated nucleotides at most of the genomic fusion junctions, suggesting that a nonhomologous end‐joining repair mechanism was involved in the t(11;16).

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

Rapid identification of BCR/ABL1-like acute lymphoblastic leukaemia patients using a predictive statistical model based on quantitative real time-polymerase chain reaction: clinical, prognostic and therapeutic implications

BCR/ABL1‐like acute lymphoblastic leukaemia (ALL) is a subgroup of B‐lineage acute lymphoblastic leukaemia that occurs within cases without recurrent molecular rearrangements. Gene expression profiling (GEP) can identify these cases but it is expensive and not widely available. Using GEP, we identified 10 genes specifically overexpressed by BCR/ABL1‐like ALL cases and used their expression values – assessed by quantitative real time‐polymerase chain reaction (Q‐RT‐PCR) in 26 BCR/ABL1‐like and 26 non‐BCR/ABL1‐like cases to build a statistical “BCR/ABL1‐like predictor”, for the identification of BCR/ABL1‐like cases. By screening 142 B‐lineage ALL patients with the “BCR/ABL1‐like predictor”, we identified 28/142 BCR/ABL1‐like patients (19·7%). Overall, BCR/ABL1‐like cases were enriched in JAK/STAT mutations (P < 0·001), IKZF1 deletions (P < 0·001) and rearrangements involving cytokine receptors and tyrosine kinases (P = 0·001), thus corroborating the validity of the prediction. Clinically, the BCR/ABL1‐like cases identified by the BCR/ABL1‐like predictor achieved a lower rate of complete remission (P = 0·014) and a worse event‐free survival (P = 0·0009) compared to non‐BCR/ABL1‐like ALL. Consistently, primary cells from BCR/ABL1‐like cases responded in vitro to ponatinib. We propose a simple tool based on Q‐RT‐PCR and a statistical model that is capable of easily, quickly and reliably identifying BCR/ABL1‐like ALL cases at diagnosis.

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.