Large-scale shRNA screens to identify novel combination therapies for the treatment of cancer BIOS 6660 Mark A. Gregory, Ph.D Research Instructor DeGregori Lab BIOS 6660 Lecture: shRNA synthetic lethal screening Overview: 1) Biological problem: Chronic Myeloid Leukemia (CML) -finding the right genes to target to improve CML therapy 2) Approach: large-scale shRNA synthetic lethal screening 3) How shRNA screen data can be translated into a therapy 4) New biological problem: Acute Myeloid Leukemia (AML) -finding the right genes to target to improve AML therapy Chronic Myeloid Leukemia (CML) • Chronic myeloid leukemia (CML) is a myeloproliferative disorder of hematopoietic stem cell origin that is characterized by the t(9;22) translocation, which gives rise to a shortened chromosome 22, the “Philadelphia chromosome” (Ph). • This results in a novel fusion protein, p210 Bcr-Abl, that has constitutive tyrosine kinase activity and is causative in the disease. • CML is a triphasic disease, beginning with a relatively stable chronic phase that lasts on average 4-5 years, progressing into an accelerated phase (6-18 months), and terminating in fatal blast crisis (~6 months). • Imatinib mesylate (Gleevec is a small-molecule Bcr-Abl kinase inhibitor that has revolutionized the treatment of CML. Mechanism of action of imatinib Effector Bcr-Abl substrate ATP P Y PPP Y Effector Bcr-Abl Imatinib substrate Y substrate Y proliferation survival Imatinib is an effective treatment for Bcr-Abl+ leukemia, but it is not a cure Imatinib induces remarkable hematological and cytogenetic responses in chronic phase CML patients However, imatinib fails to completely eradicate Bcr-Abl+ leukemic cells (Bcr-Abl remains detectable in >95% of responding patients) CML patients often develop resistance to imatinib through mutation or amplification of Bcr-Abl Advanced phase CML (blast crisis) and Bcr-Abl+ acute lymphoblastic leukemia (ALL) are poorly responsive to imatinib therapy A second generation of more potent Bcr-Abl inhibitors has been developed (nilotinib, dasatinib) but they do not solve these problems Our problem: Bcr-Abl inhibition alone is insufficient to effectively eleminate leukemic cells in CML and in BcrAbl+ ALL Our hypothesis: Targeting an additional gene product may potentiate the efficacy of Bcr-Abl inhibitors in eliminating Bcr-Abl+ cells and lead to complete eradication of the disease How do we find such genes? Our approach: Design and perform unbiased largescale loss-of-function screen (synthetic lethal) utilizing an shRNA library to identify gene targets that, when inhibited, potentiate the efficacy of imatinib in killing CML cells Synthetic Lethality Concept A B Alive A B Alive A B Dead Gene A: Bcr-Abl Gene B: unknown (screen for using RNAi) Harnessing the power of RNAi http://www.gene-quantification.de shRNA X gene X Our RNAi Synthetic Lethal Screen on CML * 3X (triplicate cultures) puro K562 CML cells 3X (triplicate cultures) Imatinib (Bcr-Abl inhibitor) * Genome-wide Library contains 4-10 shRNA’s per gene, targeting all human genes = 200,000 different shRNAs. Delivered to cells using lentivirus. Plasmid used to make shRNA containing virus 21bp siRNA sequences Lentiviral Packaging Element Polylinker for cloning PuromycinResistance for selection in mammalian cells 5’ and 3’ LTRs for viral transcription control Ori and AmpRes for replication and expansion in E. coli RNA Product (shRNA) TRC = The RNA Consortium http://www.sigmaaldrich.com Lentiviral transduction delivers a single shRNA to every cell (e.g Bcr-Abl) shRNA inhibits gene in pathway Deep sequencing is used to quantify shRNA’s shRNA counts Deep Sequencing Data Control Treatment shRNA1 80 90 shRNA2 40 40 shRNA3 100 100 shRNA4 100 0 shRNA5 60 50 shRNA6 60 80 = strong synthetic lethal What did we find in CML screen? identified shRNA’s targeting 146 genes as under-represented >16-fold (confidence interval > 99.5%) in imatinib-treated vs. untreated cells ie. these shRNA’s cooperated with imatinib in CML cell killing. The genes these shRNA’s target = SLIM’s : Synthetic Lethal with Imatinib Mesylate Major SLIM pathway: Noncanonical Wnt/Ca2+ pathway Almost every gene in this pathway came up in screen with one or more shRNA as being Synthetic Lethal with Imatinib Mesylate Wnt5a Fzd Cyclosporin A (CsA) G prot PDE PLC DAG IP3 Ca2+ CaMKII Calm PKC Calcn NF-kB NFAT cytokines IL-4 AP-1 nucleus The calcineurin inhibitor CsA cooperates with imatinib in killing K562 blast crisis CML cells in vitro CsA (0, 1, 2.5, or 5 µM) 0 0.1 1.0 • CsA potently inhibits NFAT activity in CML cells µM imatinib after72 hr treatment Combined therapy with CsA and Bcr-Abl inhibitor dasatinib leads to prolonged survival in a mouse model of Bcr-Abl+ leukemia Gregory et al., Cancer Cell (2010) These data eventually led to a Phase 1 clinical trial exploring Dasatinib + CsA ClinicalTrials.gov Identifier: NCT01426334 Dasatinib and Cyclosporine in Treating Patients With Chronic Myelogenous Leukemia Refractory or Intolerant to Imatinib Mesylate Official Title ICMJ: Exploiting Synergy in Chronic Myelogenous Leukemia: A Phase Ib Evaluation of Dasatinib Plus Cyclosporine in Patients With Ph+ Leukemia (ESCAPE1b) Brief Summary : This phase I trial studies the side effects and the best way to give dasatinib and cyclosporine in treating patients with chronic myelogenous leukemia (CML) refractory or intolerant to imatinib mesylate. Dasatinib may stop the growth of cancer cells by blocking some of the enzymes needed for cell growth. Cyclosporine may help dasatinib work better by making cancer cells more sensitive to the drug. Giving dasatinib together with cyclosporine may be an effective treatment for CML. Demonstrates how a functional genomics screen can identify a therapeutic strategy that rapidly translates to the clinic for potential patient benefit New biological problem: Acute Myeloid Leukemia Acute myeloid leukemia is a heterogeneous disease characterized by the uncontrolled proliferation of hematopoietic progenitor cells An estimated 13,780 new cases of AML were diagnosed in U.S. in 2011 and there were >10,000 estimated deaths from AML Response to chemotherapy is poor and most patients will die of their disease (only 40% of patients <60 yo and only 10% of older patients will remain in remission >5 years) We are desparate for better therapies Confronting a Broad Spectrum of Diseases With Diverse Outcomes Comparison of Diseases by Survival Rate, Age of Onset & Incidence NHL MM CLL Average Age of Onset AML CML MDS MPD Incidence HL 58,000 ALL 4,300 SEER database, scientific literature Median 5-year Survival Rate Targeting AML: FLT3 FLT3 (fms-like tyrosine kinase 3) is receptor tyrosine kinase expressed on hematopoietic progenitor cells Activating mutations of FLT3 (ITD and TK domain) are present in 30-40% of AMLs and are associated with aggressive disease and poor prognosis FLT3 is a potentially promising therapeutic target for treatment of AML FLT3 signaling Promotes growth, proliferation and survival FLT3 inhibitors fail to achieve durable remissions in AML In clinical trials, FLT3 inhibitors (e.g. CEP-701, AC220) show significant anti-leukemic activity in FLT3 mutated (FLT3MT) AML However, most of the responses consisted of a clearance of peripheral leukemic blasts and major reductions in bone marrow blasts were not typically achieved Responses were transient with patients blasts returning within a few weeks to a few months Problem: FLT3 inhibition alone is insufficient to effectively eleminate leukemic cells in FLT3MT AML Our hypothesis: Targeting additional genes may potentiate the efficacy of FLT3 inhibitors in eliminating FLT3 leukemic cells and lead to complete eradication of the disease Our approach: Large-scale shRNA synthetic lethal screen Our RNAi Synthetic Lethal Screen on AML * 3X (triplicate cultures) puro Molm AML cells 3X (triplicate cultures) CEP-701 (FLT3 inhibitor) * Genome-wide Library contains 4-10 shRNA’s per gene, targeting all human genes = 200,000 different shRNAs. Delivered to cells using lentivirus. Give sequencing datasets to BIOS 6660 students for Bioinformatics Analysis. Ask them to identify genes that are “SLAMs” – Synthetic Lethal in Acute Myeloid Leukemia. Align sequences to shRNA Library Accounting for: Pathways Analysis (Ingenuity, DAVID, KEGG) Aik Choon Tan Jihye Kim • Relative shRNA representation • Correlation between distinct shRNAs targeting the same gene • Replication across experiments (typically 3 Vehicle, 3 Treatment) What are we looking for in the final analysis? 1) A list of the top genes identified as SLAMs 2) A list of the top SLAM pathways 3) An idea for a potentially promising combination therapy, i.e. FLT3 inhibitor + drug X that will more effectively treat or cure AML. Publications from our group employing synthetic lethal screening Alvarez-Calderon F, Gregory MA, and DeGregori J. Using functional genomics to overcome therapeutic resistance in hematological malignancies. Immunol Res. 2013 Mar;55(1-3):100-15. Gregory MA, Phang TL, Neviani P, Alvarez-Calderon F, Eide CA, O'Hare T, Zaberezhnyy V, Williams RT, Druker BJ, Perrotti D, and Degregori J. Wnt/Ca2+/NFAT signaling maintains survival of Ph+ leukemia cells upon inhibition of Bcr-Abl. Cancer Cell. 2010 Jul 13;18(1):74-87. Casás-Selves M, Kim J, Zhang Z, Helfrich BA, Gao D, Porter CC, Scarborough HA, Bunn PA Jr, Chan DC, Tan AC, and Degregori J. Tankyrase and the Canonical Wnt Pathway Protect Lung Cancer Cells from EGFR Inhibition. Cancer Res. 2012 Aug 15;72(16):4154-64. Porter CC, Kim J, Fosmire S, Gearheart CM, van Linden A, Baturin D, Zaberezhnyy V, Patel PR, Gao D, Tan AC, and DeGregori J. Integrated genomic analyses identify WEE1 as a critical mediator of cell fate and a novel therapeutic target in acute myeloid leukemia. Leukemia. 2012 Jun;26(6):1266-76. Sullivan KD, Padilla-Just N, Henry RE, Porter CC, Kim J, Tentler JJ, Eckhardt SG, Tan AC, DeGregori J, and Espinosa JM. ATM and MET kinases are synthetic lethal with nongenotoxic activation of p53. Nat Chem Biol. 2012 Jul;8(7):646-54. doi: 10.1038/nchembio.965.