Large-scale shRNA screens to identify novel combination

therapies for the treatment of cancer

Mark A. Gregory, Ph.D

Research InstructorDeGregori 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-AblATP substrateP P P Y

Effector

Effector

Mechanism of action of imatinib

substrate

YP

substrateY

Bcr-AblImatinib substrate

Y

proliferationsurvival

growth arrestapoptosis

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

A B

A B

A B

Alive

Alive

Dead

Gene A: Bcr-Abl Gene B: unknown (screen for using RNAi)

Harnessing the power of RNAi

SYNTHETIC LETHAL

SCREENING

http://www.gene-quantification.deshRNA X gene X

Our RNAi Synthetic Lethal Screen on CML

Imatinib(Bcr-Abl inhibitor)

K562CML 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)

3X (triplicate cultures)

http://www.sigmaaldrich.com

RNA Product(shRNA)

Polylinker for cloning

PuromycinResistanc

e for selection in mammalian cells

Ori and AmpRes for replication and expansion in E. coli

21bp siRNAsequences

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

S = SYNTHETIC LETHAL

shRNA inhibitsgene in pathway

VehicleSSSSS

Inhibitor S

S S

(e.g Bcr-Abl)

shRNA1

shRNA2

shRNA3

shRNA4

shRNA5

shRNA6

Control Treatment

80 90

40 40

100 100

100 0

60 50

60 80

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

PLCPDE

Fzd

Calcn

DAG

Major SLIM pathway: Noncanonical Wnt/Ca2+ pathway

NFAT

IP3Ca2+

Calm

IL-4cytokines nucleus

NF-kB AP-1

Cyclosporin A (CsA)

Almost every gene in this pathway came up in screen with one or more shRNA as beingSynthetic 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

0.10 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 exploringDasatinib + CsA

Demonstrates how a functional genomics screen can identifya 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

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

Ave

rage

Age

of O

nset

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 (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

CEP-701(FLT3 inhibitor)

MolmAML 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)

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 TanJihye Kim VALIDATION

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 screeningAlvarez-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.