Identifikation neuer Onkogene mit funktioneller Relevanz für die Pathogenese peripherer T-Zell Lymphome
Final Report Abstract
There is a pressing need for methods to define the functional relevance of genetic alterations identified by next-generation sequencing of cancer specimens. We developed new approaches to efficiently construct full-length cDNA libraries from small amounts of total RNA, screen for transforming and resistance phenotypes, and deconvolute by next-generation sequencing. Using this platform, we screened a panel of cDNA libraries from primary specimens and cell lines in cytokine-dependent unmodified and modified Ba/F3 cells. We demonstrate that cDNA library-based screening can efficiently identify DNA and RNA alterations that confer either cytokine-independent proliferation or resistance to targeted inhibitors, including RNA alterations and intergenic fusions. Using barcoded next-generation sequencing, we can simultaneously deconvoluted hundreds of cytokine-independent clones from a variety of different tumor libraries. This approach identified multiple gain-of-function alleles, including an ATIC-ALK fusion transcript from a patient’s T-cell lymphoma sample, representing a small inversion on chromosome 2 that was cryptic on karyotyping. This approach has broad applicability for identifying 9transcripts that confer proliferation, resistance and other phenotypes in-vitro and potentially in-vivo. We are currently performing large-scale cDNA screenings in collaboration with the Cancer Cell Line 25Encyclopedia (CCLE, www.broadinstitute.org/ccle/home). Enzymatic inhibitors of Janus kinase 2 (JAK2) are in clinical development for the treatment of myeloproliferative neoplasms (MPNs), B cell acute lymphoblastic leukemia (B-ALL) with rearrangements of the cytokine receptor subunit cytokine receptor–like factor 2 (CRLF2), and other tumors with constitutive JAK2 signaling. In this study, we identify G935R, Y931C, and E864K mutations within the JAK2 kinase domain that confer resistance across a panel of JAK inhibitors, whether present in cis with JAK2 V617F (observed in MPNs) or JAK2 R683G (observed in B-ALL). G935R, Y931C, and E864K do not reduce the sensitivity of JAK2-dependent cells to inhibitors of heat shock protein 90 (HSP90), which promote the degradation of both wild-type and mutant JAK2. HSP90 inhibitors were 100–1,000-fold more potent against CRLF2-rearranged B-ALL cells, which correlated with JAK2 degradation and more extensive blockade of JAK2/STAT5, MAP kinase, and AKT signaling. In addition, the HSP90 inhibitor AUY922 prolonged survival of mice xenografted with primary human CRLF2-rearranged B-ALL further than an enzymatic JAK2 inhibitor. Thus, HSP90 is a promising therapeutic target in JAK2-driven cancers, including 11those with genetic resistance to JAK enzymatic inhibitors. Based on these results a phase 2 clinical trial of HSP90 inhibition in relapsed ALL is currently being initiated. The relative timing of genetic alterations that contribute to lymphoma remains unknown. We analyzed a donor–recipient pair who both developed grade 2/3A follicular lymphoma 7 years after allogeneic transplantation and donor lymphocyte infusions. Both patients harbored identical BCL2/IGH rearrangements also present in 1 in 2,000 cells in the donor lymphocyte infusion, and the same V(D)J rearrangement, which underwent somatic hypermutation both before and after clonal divergence. Exome sequencing of both follicular lymphomas identified 15 shared mutations, of which 14 (including alterations in EP300 and KLHL6) were recovered from the donor lymphocyte infusion by ultra-deep sequencing (average read coverage, 361,723), indicating acquisition at least 7 years before clinical presentation. Six additional mutations were present in only one follicular lymphoma and not the donor lymphocyte infusion, including an ARID1A premature stop, indicating later acquisition during clonal divergence. Thus, ultrasensitive sequencing can map clonal evolution within rare subpopulations during human lymphomagenesis in-vivo. For the first time, we define the molecular ontogeny of follicular lymphoma during clonal evolution in-vivo. By using ultrasensitive mutation detection, we mapped the time-course of somatic alterations after passage of a malignant ancestor by hematopoietic cell 12transplantation. Based on these results and follow-up data, we proposed a modified model of lymphomagenesis that accounts for mutation ontogeny and raises the possibility, that somatic 13alterations in early hematopoietic progenitors contribute to disease biology.
Publications
- BCL2 suppresses PARP1 function and non‐apoptotic cell death. Cancer Res. 2012 Aug 15;72(16):4193‐203
Dutta C, Day T, Kopp N, van Bogedom D, Davids M, Ryan J, Bird L, Kommajosyula N, Weigert O, et al.
- Differences in signaling through the B-cell leukemia oncoprotein CRLF2 in response to TSLP and through mutant JAK2. Blood. 2012 Oct 4;120(14):2853-63
van Bodegom D, Zhong J, Kopp N, Dutta C, Kim MS, Bird L, Weigert O, et al.
- Genetic resistance to JAK2 enzymatic inhibitors is overcome by HSP90 inhibition. J. Exp. Med., Feb 2012; 209: 259-273
Weigert O, Lane AA, Bird L, Kopp N, et al.
- Molecular Ontogeny of Donor‐Derived Follicular Lymphomas Occurring after Hematopoietic Cell Transplantation. Cancer Discovery, Jan 2012; 2: 47-55
Weigert O, Kopp N, Lane AA, Yoda A, et al.
- The evolving contribution of hematopoietic progenitor cells to lymphomagenesis. Blood. 2012 Aug 6. [Epub ahead of print]
Weigert O, Weinstock DM
