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.