Drug is currently approved for Ph+ CML and Ph+ ALL.

Scientists have pinpointed a molecular pathway that may help identify subsets of renal cell carcinoma (RCC) patients who would benefit from Src inhibitors likes dasatinib. They say an HIF-regulated VHL-PTP1B-Src signaling pathway determines the sensitivity of RCC.

Collaborators in the U.S. and U.K., led by George V. Thomas, M.D., at Oregon Health and Science University’s (OHSU) Knight Cancer Institute, report their results in Science Translational Medicine. The paper is titled “A HIF-Regulated VHL-PTP1B-Src Signaling Axis Identifies a Therapeutic Target in Renal Cell Carcinoma.”

Dasatinib is being developed by Bristol-Myers Squibb. It is currently approved for Philadelphia chromosome positive (Ph+) chronic myeloid leukemia and Ph+ acute lymphoblastic leukemia and marketed as Sprycel.

Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene is the most prevalent driver mutation in RCC, accounting for about 60% of all RCC tumors and occurring primarily in the clear cell subtype, the researchers state. Previous work has suggested that VHL loss stabilizes hypoxia inducible factor-1a (HIF-1a) and HIF-2a, leading to increased expression of HIF-responsive genes including VEGF-A, DGFR-B, and TGF-A. HIF-dependent gene expression is further elevated by mammalian target of rapamycin (mTOR).

These findings have collectively provided a number of avenues for RCC therapeutic development, yet while drugs that target VEGF and mTOR do show clinical activity in patients with metastatic RCC, responses are often variable and short-lived, the OHSU researchers write. Moreover, there remains a lack of treatment options for the 40% or so of patients with VHL-positive RCC.

To try and identify signaling networks that are differentially expressed in RCC subgroups and may yield new subtype-specific drug targets, Dr. Thomas and colleagues carried out quantitative phosphoproteomics on a clear cell carcinoma cell line that retains VHL protein expression and its isogenic subline in which VHL has been knocked down using an shRNA. The results suggested that VHL expression is a key determinant of Src kinase activity; a promising finding given that Src levels and its activity have previously been implicated in cancer development.

The researchers then Interrogated a human RCC tissue microarray with samples from over 200 RCC patients. They found that high levels of Src expression and activity correlated with reduced survival. Multivariate analyses suggested that high Src levels were, in fact, an independent predictor of poor survival.

They then treated two different pairs of VHL-expressing and matched VHL-knockout cell lines with the Src inhibitor dasatinib. The drug reduced proliferation of the two wild-type cell lines but not their VHL-knockout counterparts. In the wild-type cells dasatinib caused cells to arrest in the growth phase and decreased DNA synthesis. Further in vitro studies confirmed that dasatinib’s activity was at least in part related to the inhibition of Src kinase activation and the phosphorylation of Src substrates.

When these cancer cell lines were implanted in nude mice, daily treatment with dasatinib significantly reduced the growth of VHL-expressing wild-type tumors but had no effect on the growth of VHL knockout tumors.

To confirm that the tumor growth inhibitor effects of dasatinib were indeed related to Src activity, the team then evaluated the effects of the drugs on cancer cells in which Src activity was inhibited using a different shRNA. VHL-expressing RCC cells in which Src activity was blocked were indeed resistant to dasatinib therapy, an effect that could be reversed by separately treating them with chicken Src, which isn’t susceptible to the shRNA used to knock down the human Src.

Importantly, treating RCC cell lines with imatinib, a drug that inhibits ABL, PDGFR, and c-KIT but not Src, had no effect on the proliferation of the VHL wild-type or VHL-knockdown cells. Saracatinib, on the other hand, which is a structurally unrelated Src inhibitor, did repress proliferation of the wild-type cells but not the VHL-knockdown cells.

VHL negatively regulates HIF, and further tests showed that forcing the expression of HIF in RCC cells with wild-type VHL diminished Src signaling output by repressing transcription of the Src activator protein tyrosine phosphatase 1B (PTP1B), which effectively resulted in dasatinib resistance. Conversely, ectopic expression of VHL in VHL-null RCC cells resulted in an increase in Src as well as the phosphorylation and activation of its downstream targets in comparison with parental cells.

The authors hypothesized that the ability of constitutively stable HIF mutants to promote dasatinib resistance and repress Src signaling output suggests that HIF may repress an activator of Src. Consistent with this, the researchers found that levels of the  PTP1B protein and expression of its mRNA was consistently lower in VHL knockout  RCC cells or cells that ectopically expressed constitutively stable HIF.

The identification of an HIF-regulated VHL-PTP1B-Src signaling axis in RCC cell lines provided additional markers for analyzing the presence of this pathway in RCC patients. The authors constructed a tissue microarray from a second cohort of 131 patients with RCC and performed immunohistochemistry for VHL, HIF-2a (which is the primary driver in VHL-null RCC), Src, and PTP1B. In agreement with the initial RCC clinical data set the new findings again revealed a positive correlation between VHL and Src. In fact, only 8% of VHL-negative tumors had strong Src expression, whereas 58% of tumors with high VHL expression also had strong Src expression, suggesting a correlative relationship between VHL and Src. Conversely, the relationship between VHL and HIF-2a revealed a significant negative correlation.

Also in agreement with the in vitro findings, analyses of the human tumors showed that PTP1B positively correlated with VHL but negatively correlated with HIF-2a , suggesting that tumors with VHL loss or HIF-2a overexpression may have reduced PTP1B expression. “Indeed, 28.6% of the patients were VHL strong/Src strong, representing the potential candidates for a prospective Phase II clinical trial with dasatinib,” the authors remark.

Interestingly, the same parameters could be applied to other cancers to predict sensitivity to dasatinib. Using a clinical data set of transitional cell carcinomas of the bladder, the researchers found the same correlation between VHL, Src, HIF-2a, and PTP1B.

“Together, these findings suggest that the immunophenotype of the VHL-PTP1B-Src signaling axis comprises a signature that not only defines a biologically distinct subgroup of RCC that may benefit from dasatinib or similar Src inhibitors but also points to a wider clinical applicability for these predictive markers in identifying sensitivity to Src inhibitors,” the authors conclude.

“Our data suggests that stratifying RCC patients by profiling for expression of VHL and Src as well as downstream effector molecules, may identify patients likely to respond to Src inhibitors in future RCC clinical trials.”

Src is itself rarely mutated in cancers, and the OHSU team’s data suggest that PTP1B acts as one of the drivers used by tumor cells to amplify Src kinase activity. “The ability of PTP1B knockdown to confer resistance to dasatinib suggests that PTP1B may augment Src signaling in RCC cells by channelling inputs from upstream oncogenes including Ras,” they suggest. “Our findings suggest that Src inhibition may represent a rational treatment option in renal cancers that retain VHL protein expression.”

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