Growth Suppressive Effects of  Inhibitors of STAT3, Syk, and Braf on Chronic Lymphocytic Leukemia:

Superior Efficacy of  STAT3 Inhibitor

Melissa ShadoinDoane CollegeUNMC Department Genetics Cell Biology and AnatomyAugust 3, 2015

Outline• Background on CLL• Hypothesis and Rationale• Methodology• Results• Summary• Future Studies• Acknowledgement

Chronic Lymphocytic Leukemia (CLL)

• Indolent B cell malignancy which is characterized by CD19+CD5+CD23+ cells accumulated in peripheral blood, bone marrow, and lymph nodes.

• CLL represents the most common adult leukemia in western countries.

• Incidents increase with age with median age of 65.

• Approximately 10,000 cases every year in the United States.

Cytomorphology of CLL cells

Current Research on CLL• CLL was considered as tumor of long lived resting B cells with impaired

apoptosis.

• CLL patients have a very small actively proliferating population which resides in proliferating centers in the lymph node and bone marrow.

• These proliferating centers create tumor microenvironment (TME) which provides stimuli for the CLL cells’ survival and proliferation.

• Emerging evidence including our laboratory findings suggest that TME activates several pathways in the proliferating CLL cells. This in turn results in heterogeneous clinical outcome.

• We and others have also found that SYK, BTK, STAT3 and Braf are constitutively activated in proliferating CLL cells.

• Targeting these molecules using their specific small molecule inhibitors might help us in developing an effective therapy for CLL.

Tumor Microenvironment of CLL cells

RationaleThere are several reports which have shown that SYK mediated MAPK signaling is hyperactivated in CLL cells.

Stimulation of B-cell Receptor(BCR) of CLL cells leads to hyperactivation of STAT3 signaling pathway.

Several mutations has been identified in molecules leading to deregulated MAPK signaling including BRAF, RAF and Ras.

HypothesisTargeting Syk, STAT3, and MAPK signaling pathways will decrease survival and proliferation of CLL cells.

MethodologySyk mediated signaling pathway: Syk inhibitor (R788)

MAPK signaling pathway: BRAF inhibitor (AZ628)

STAT3 signaling pathway: STAT3 inhibitor (STATIC)

Treated primary CLL cells from patients and Mec-1 CLL cell line with four different doses for 24 and 48 hours To study the efficacy of drugs we performed Microculture Tetrazolium (MTT) assay to measure cell viability.Annexin V staining to measure the rate of apoptosis.

STAT3 inhibitor has a higher efficacy than Syk and Braf inhibitors.

24 Hours 48 Hours

Mec-1

CLL cells

* p < .05

n=11

Efficacy of Syk and Braf inhibitor increased when combined with 5 µM stat3 inhibitor

24 Hours 48 Hours

Mec-1

CLL cells

* p < .05

n=3

Apoptosis measured via Annexin V• Annexins are calcium-dependent phospholipid-binding

proteins that preferably bind to phosphatidyl serine.

Apoptosis for 10µM concentration of inhibitors

21%23%

93% 32% 29%

Cells Only DMSO

Stat3 Syk Braf

Syk and Braf combined with Stat3 inhibitor increase apoptosis

32%

29% 68%

94%

Syk Only Syk with 5µM Stat3

Braf Only Braf with 5µM Stat3

Summary• Although all three inhibitors suppressed the

survival and proliferation of CLL cells, STAT3 inhibitor has higher efficacy compared to Syk and Braf inhibitor.

• STAT3 inhibitor has synergistic inhibitory effect on CLL cell survival in combination with Syk and Braf inhibitors.

Future Studies• Test the effect of STAT3, Syk and BRAF inhibitors on their

associated signaling pathways.

• Test the efficacy of these drugs in vivo using xenograft mouse model and Mec-1 CLL cells.

• To study the role these signaling pathways in tumor microenvironment using co-culture studies, i.e. culturing stromal and CLL cells together for varying length of time.

AcknowledgementsJoshi Laboratory

-- Dr. Shantaram Joshi, Ph.D• Dr. Nagendra Chaturvedi, Ph.D• Ashima Shukla, B.S.• Matthew Kling, M.S.• Garrett Sutton, B.S.• Michael Pitner, B.S.

INBRE Program– Dr. James Turpen, Ph.D– Dr. William Chaney, Ph.D– Saralyn Fisher

Funding From: – Grant Number P20 GM103427 from NIGMS, NIH– Lymphoma Research Foundation, New York, NY, ; UNMC College of Medicine

Deans Research & development Funds; GCBA Faculty Pilot Project Funds.– UNMC Tobacco Research Funds.