Shuo-Chieh Wu

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.

Mutations in G protein β subunits promote transformation and kinase inhibitor resistance

Activating mutations in genes encoding G protein α (Gα) subunits occur in 4–5% of all human cancers, but oncogenic alterations in Gβ subunits have not been defined. Here we demonstrate that recurrent mutations in the Gβ proteins GNB1 and GNB2 confer cytokine-independent growth and activate canonical G protein signaling. Multiple mutations in GNB1 affect the protein interface that binds Gα subunits as well as downstream effectors and disrupt Gα interactions with the Gβγ dimer. Different mutations in Gβ proteins clustered partly on the basis of lineage; for example, all 11 GNB1 K57 mutations were in myeloid neoplasms, and seven of eight GNB1 I80 mutations were in B cell neoplasms. Expression of patient-derived GNB1 variants in Cdkn2a-deficient mouse bone marrow followed by transplantation resulted in either myeloid or B cell malignancies. In vivo treatment with the dual PI3K-mTOR inhibitor BEZ235 suppressed GNB1-induced signaling and markedly increased survival. In several human tumors, mutations in the gene encoding GNB1 co-occurred with oncogenic kinase alterations, including the BCR-ABL fusion protein, the V617F substitution in JAK2 and the V600K substitution in BRAF. Coexpression of patient-derived GNB1 variants with these mutant kinases resulted in inhibitor resistance in each context. Thus, GNB1 and GNB2 alterations confer transformed and resistance phenotypes across a range of human tumors and may be targetable with inhibitors of G protein signaling.

Abstract PR07: Novel oncogenic mutations in the beta subunit of heteromeric G-proteins identified by functional cDNA library screening.

Although next-generation sequencing can delineate the genetic alterations within a primary tumor specimen, it can be difficult to distinguish the small number of driver mutations from the large number of passenger mutations. To overcome this issue, we developed a system for identifying oncogenic alterations directly from tumor cells. In this system, retroviral cDNA libraries built from cancer cell lines and directly from primary cancer samples are transduced into BaF3 cells, an IL3-dependent B cell line. Transformation by oncogenes promotes IL3-independent survival, allowing for the isolation of individual transformed clones and sequencing of the integrated cDNA. In the past, we identified CRLF2 as a novel oncogene in acute lymphoblastic leukemia (Yoda et al. PNAS 2010). We have improved the method and demonstrated 100% sensitivity for isolating well-characterized oncogenes, including EGFR, HER2, RAS and ALK (Shindoh et al. PLoS One 2012). Recently, we isolated a mutated GNB1 K89E allele from a cDNA library generated from a primary blastic plasmacytoid dendritic cell neoplasm (BPDCN). BPDCN is a rare and aggressive leukemia with a dismal prognosis. GNB1 encodes the beta subunit of the heterotrimeric G-protein, a binding complex that transduces signals from G-protein coupled receptors to multiple downstream pathways. Gain-of-function mutations have been reported in alpha subunits of the G-protein, however, the contributions of beta subunits to cancer remains undefined. To investigate downstream signaling from GNB1 K89E, we performed gene expression profiling and mass spectrometry (MS)-based phosphoproteomics and found significant activation of RAS/MAPK and PI3K/AKT pathways in GNB1 K89E-expressing cells compared to isogenic cells expressing wild-type GNB1. To target GNB1 K89E signaling, we screened kinase inhibitors using a multiplex panel of small molecules and found selective sensitivity of GNB1 K89E cells to MEK and pan-PI3-kinase inhibitors. Next, we transduced GNB1 alleles into bone marrow cells from Cdkn2a-deficient mice and transplanted into wild-type recipient mice. Within 4 months after transplantation, all mice (n=10) that received bone marrow transduced with GNB1 K89E developed a lethal dendritic cell malignancy, confirming the transforming effects of GNB1 K89E in vivo. A search of published cancer mutations identified four cases with GNB1 I80T/N in chronic lymphocytic leukemia or B-cell lymphomas, five cases with GNB1 K57E/T in myeloid malignancies, one case of GNB1 K89E in acute lymphoblastic leukemia, and two cases with GNB2 M101T/V in ovarian cancer. All of these alleles promoted GM-CSF-independent growth in human TF1 cells. Interestingly, the mutated codons are all located on the GNB1 molecular surface that is critical for interactions between GNB1 and both alpha subunits and downstream effectors. Immunoprecipitation followed by MS demonstrated that GNB1 K89E and I80T mutants failed to bind inhibitory G alpha subunits GNAI2 and GNAI3 as well as GNA11 that are bound by wild-type GNB1. Thus, gain-of-function mutations in G-protein beta subunits occur across a broad range of malignancies, can drive in vivo transformation, and activate targetable downstream kinases by modifying essential interactions with partner proteins. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):PR07. Citation Format: Akinori Yoda, Guillaume Adelmant, Nobuaki Shindoh, Bjoern Chapuy, Yuka Yoda, Oliver Weigert, Nadja Kopp, Shuo-Chieh Wu, Sunhee S. Kim, Huiyun Liu, Trevor Tivey, Jeffrey W. Tyner, Jason Gotlib, Michael W. Deininger, Shannon Turley, Jarrod A. Marto, Andrew A. Lane, David M. Weinstock. Novel oncogenic mutations in the beta subunit of heteromeric G-proteins identified by functional cDNA library screening. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr PR07.

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.

Abstract 433: Triplication of HMGN1 promotes B cell acute lymphoblastic leukemia (B-ALL) through suppression of H3K27me3

Our goal is to identify oncogenic loci in regions of recurrent DNA copy number alterations in cancer. Constitutional trisomy 21 (Down syndrome) carries a 20-fold increased risk of B-ALL, and chr.21 gains are the most common acquired aneuploidy in B-ALL. Interstitial amplification in the chr.21q22 region (iAMP21) is also a recurrent finding in B-ALL and carries a poor prognosis. However, the gene(s) on chr.21 responsible for this association remain unclear. We studied the Ts1Rhr mouse, which carries germline triplication of 31 genes homologous to human chr.21q22. Chr.21q22 triplication was sufficient to promote B cell autonomous self-renewal and maturation defects, and cooperated with BCR-ABL or CRLF2 with JAK2 R683G to accelerate leukemogenesis. Chr.21q22 triplication also resulted in histone H3K27 hypomethylation at gene promoters, and the expression signature of triplicated B cells was enriched for genes targeted by polycomb repressor complex 2 (PRC2), which trimethylates H3K27. Thus, chr.21q22 triplication may deregulate B cell development by causing H3K27 hypomethylation at genes critical for progenitor cell growth. In support of this hypothesis, pharmacologic inhibition of PRC2 function was sufficient to confer self-renewal in wild-type B cells, while inhibition of H3K27 demethylases blocked self-renewal induced by chr.21q22 triplication. In three independent B-ALL cohorts, PRC2/H3K27 gene signatures distinguished leukemias with +21 from those without, validating the same biology in human disease. One of the 31 triplicated genes, HMGN1, encodes a nucleosome binding protein known to modulate chromatin structure and facilitate transcriptional activation. When we overexpressed HMGN1 in BaF3 proB cells, H3K27me3 decreased proportionally to the level of overexpression. We next knocked down each of the 31 triplicated genes with lentivirally-expressed shRNAs (5 per gene) and assessed the effects on growth of Ts1Rhr and wild-type primary B cells. Strikingly, Hmgn1 was the top scoring gene and all 5 hairpins targeting Hmgn1 were depleted in the assay. Finally, we studied transgenic mice (HMGN1_OE) that overexpress human HMGN1 (∼2-fold total overexpression). HMGN1_OE mice had a defect in B cell maturation, increased proB colony forming capacity, and a transcriptional signature overlapping with that of triplication of all 31 Ts1Rhr genes. In a bone marrow transplant model driven by BCR-ABL, recipients of HMGN1_OE bone marrow developed B-ALL with decreased latency (median 33 days vs not reached) and increased penetrance (17/18 vs 4/17 mice died by 80 days; leukemia-free survival difference P Citation Format: Andrew A. Lane, Bjoern Chapuy, Charles Y. Lin, Trevor Tivey, Hubo Li, Elizabeth Townsend, Diederik van Bodegom, Tovah A. Day, Shuo-Chieh Wu, Huiyun Liu, Akinori Yoda, Gabriela Alexe, Anna Schinzel, Timothy J. Sullivan, Sebastien Malinge, Jordan Taylor, Kimberly Stegmaier, Jacob Jaffe, Michael Bustin, Geertruy te Kronnie, Shai Izraeli, Marian Harris, Kristen Stevenson, Donna Neuberg, Lewis B. Silverman, Steven E. Sallan, James E. Bradner, William C. Hahn, John D. Crispino, David Pellman, David M. Weinstock. Triplication of HMGN1 promotes B cell acute lymphoblastic leukemia (B-ALL) through suppression of H3K27me3. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 433. doi:10.1158/1538-7445.AM2014-433