Presentation on theme: "Large-scale shRNA screens to identify novel combination therapies for the treatment of cancer Mark A. Gregory, Ph.D Research Instructor DeGregori Lab BIOS."— Presentation transcript:
Large-scale shRNA screens to identify novel combination therapies for the treatment of cancer Mark A. Gregory, Ph.D Research Instructor DeGregori Lab BIOS 6660
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) 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. Chronic Myeloid Leukemia (CML)
Bcr-Abl ATP PPP Y Effector Mechanism of action of imatinib substrate Y P Y Bcr-Abl Imatinib 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 approach: Design and perform unbiased large- scale 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 Our problem: Bcr-Abl inhibition alone is insufficient to effectively eleminate leukemic cells in CML and in Bcr- Abl + 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?
Synthetic Lethality Concept AB AB A B Alive Dead Gene A: Bcr-Abl Gene B: unknown (screen for using RNAi)
http://www.gene-quantification.de shRNA X gene X
Our RNAi Synthetic Lethal Screen on CML Imatinib (Bcr-Abl inhibitor) K562 CML cells puro * * Genome-wide Library contains 4-10 shRNA’s per gene, targeting all human genes = 200,000 different shRNAs. Delivered to cells using lentivirus. 3X (triplicate cultures)
http://www.sigmaaldrich.com RNA Product (shRNA) Polylinker for cloning Puromycin Resistance for selection in mammalian cells Ori and Amp Res for replication and expansion in E. coli 21bp siRNA sequences Lentiviral Packaging Element 5’ and 3’ LTRs for viral transcription control TRC = The RNA Consortium Plasmid used to make shRNA containing virus
Lentiviral transduction delivers a single shRNA to every cell
shRNA1 shRNA2 shRNA3 shRNA4 shRNA5 shRNA6 ControlTreatment 8090 40 100 0 6050 6080 Deep Sequencing Data shRNA counts Deep sequencing is used to quantify shRNA’s = 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
PKC Wnt5a CaMKII G prot PLC PDE Fzd Calcn DAG Major SLIM pathway: Noncanonical Wnt/Ca 2+ pathway NFAT IP3 Ca 2+ Calm IL-4 cytokines nucleus NF- B AP-1 Cyclosporin A (CsA) Almost every gene in this pathway came up in screen with one or more shRNA as being Synthetic Lethal with Imatinib Mesylate
The calcineurin inhibitor CsA cooperates with imatinib in killing K562 blast crisis CML cells in vitro after72 hr treatment (0, 1, 2.5, or 5 µM) CsA potently inhibits NFAT activity in CML cells CsA 0.1 0 1.0 µM imatinib
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)
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. ClinicalTrials.gov Identifier: NCT01426334 These data eventually led to a Phase 1 clinical trial exploring Dasatinib + CsA 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 5 years) We are desparate for better therapies
Confronting a Broad Spectrum of Diseases With Diverse Outcomes SEER database, scientific literature Comparison of Diseases by Survival Rate, Age of Onset & Incidence Median 5-year Survival Rate MM NHL CLL CML MPD HL ALL Average Age of Onset 58,000 4,300 Incidence AML MDS
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 (FLT3 MT ) 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 FLT3 MT 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 CEP-701 (FLT3 inhibitor) Molm AML cells puro * * Genome-wide Library contains 4-10 shRNA’s per gene, targeting all human genes = 200,000 different shRNAs. Delivered to cells using lentivirus. 3X (triplicate cultures)
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: Relative shRNA representation Correlation between distinct shRNAs targeting the same gene Replication across experiments (typically 3 Vehicle, 3 Treatment) Pathways Analysis (Ingenuity, DAVID, KEGG) Aik Choon Tan Jihye Kim
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.