Identification of kinases required for glioblastoma stem cell growth and survival
Glioblastoma multiforme (GBM) is the most common and lethal primary malignant tumor of
the central nervous system. The median survival of patients post diagnosis is about 15
months despite the current aggressive standard of care treatment that includes surgical
resection followed by radiotherapy and chemotherapy with the DNA alkylating drug
temozolomide. Thus, there is an urgent need ... read moreto develop new therapeutic agents to improve
patient prognosis. GBM is a heterogeneous disease and a small subpopulation of tumor
cells known as cancer stem like cells (GSC) have been identified which are believed to
sustain tumor growth. These cells are refractory to current treatment and thus persist
as a disease reservoir thereby contributing to disease recurrence. In addition, GSC have
been found to preferentially reside in the hypoxic niches within the tumor and this
further contributes to the maintenance of stem cell properties and therapeutic
resistance. Therefore, it is extremely important to identify new therapeutic agents to
target these cells. Since kinases are readily druggable targets, we have carried out an
arrayed well-by-well functional lentiviral RNAi kinome screen of 3 independent patient
derived GBM stem cell lines. The screening was performed both in normoxia and hypoxia
conditions. The screen identified some genes previously known to be involved in GBM as
well as some novel hits. Overall we found 12 kinase genes which were hits common to all
the three lines screened. Interestingly, majority of the hits (~75%) were specific for
each line reflecting a high degree of heterogeneity among these cell lines and
highlighting the need for personalized therapy for GBM. In addition, there were kinase
hits that were either specific to either normoxia or hypoxia indicating differences in
kinase vulnerabilities in different microenvironments. I have further validated one of
the novel common kinase hits - YES1 (Yamaguchi sarcoma viral oncogene) which is a member
of the SRC family of tyrosine kinases. I found that YES1 is required for GBM stem cell
growth and survival using multiple shRNA, CRISPR/Cas9 constructs as well as small
molecule inhibitors. I also found that in an orthotopic xenograft model, mice implanted
with YES1 knockdown GSC had longer survival compared to the YES1 expressing controls
Finally, I have evaluated the potential of JAK kinases as therapeutic targets to inhibit
GSC. The Janus kinase (JAK)-signal transducer and activator of transcription (STAT)
signaling pathway regulates cell growth, differentiation and apoptosis in a wide variety
of tumors including GBM stem cells. Aberrant activation of the JAK/STAT3 pathway has
been implicated not only in GBM progression, but also in acquired therapy resistance and
is linked to poor patient prognosis making this pathway an attractive target for
therapy. Moreover, there are several FDA approved JAK targeting drugs available for
other diseases and this could accelerate the development of JAK inhibitors for GBM.
There are 4 JAK family members- JAK1, JAK2, JAK3 and TYK2 and determining which JAK
combination to inhibit is a key step for development of an effective therapy. I have
therefore investigated roles of individual JAK kinases to assess their relative
contributions in activating STAT3 regulating GSC growth and survival. Here I show that
JAK inhibitors strongly suppress STAT3 activation and are growth inhibitory to GSC.
Using RNAi, JAK1 was found to be dominant kinase in activating STAT3 in GSC. The results
of these studies could lead to the development of new therapies for
Thesis (Ph.D.)--Tufts University, 2017.
Submitted to the Dept. of Genetics.
Advisor: Brent Cochran.
Committee: Alain Charest, Amy Yee, and James Schwob.
Keyword: Genetics.read less