An adaptive Src-PDGFRA-Raf axis in rhabdomyosarcoma

Abstract

Alveolar rhabdomyosarcoma (aRMS) is a very aggressive sarcoma of children and young adults. Our previous studies have shown that small molecule inhibition of Pdgfra is initially very effective in an aRMS mouse model. However, slowly evolving, acquired resistance to a narrow-spectrum kinase inhibitor (imatinib) was common. We identified Src family kinases (SFKs) to be potentiators of Pdgfra in murine aRMS primary cell cultures from mouse tumors with evolved resistance in vivo in comparison to untreated cultures. Treating the resistant primary cell cultures with a combination of Pdgfra and Src inhibitors had a strong additive effect on cell viability. In Pdgfra knockout tumors, however, the Src inhibitor had no effect on tumor cell viability. Sorafenib, whose targets include not only PDGFRA but also the Src downstream target Raf, was effective at inhibiting mouse and human tumor cell growth and halted progression of mouse aRMS tumors in vivo. These results suggest that an adaptive Src-Pdgfra-Raf-Mapk axis is relevant to PDGFRA inhibition in rhabdomyosarcoma.

Figure 1. Cell autonomous resistance to the prototypic PDGFRA inhibitor, imatinib

(A) Western blot analysis showing complete reduction in phospho-Src (p-Src) and phospho-Pdgfr (p-Pdgfr) levels upon imatinib treatment in imatinib-naïve primary cell cultures whereas phospho-Pdgfr and phospho-Src were still present at detectable levels upon imatinib treatment in imatinib-resistant primary cell cultures. (B) Src family kinase inhibitors that were found to be effective against imatinib-resistant primary cell cultures in a kinase inhibitor screen. (C) Venn diagram of specificities for drugs in (B) implicates Src Family Kinases (SFK)

Figure 2. B-Raf is downstream of Src and Pdgfra in Pdgfra expressing rhabdomyosarcoma

(A) Cell viability assay for an imatinib-resistant mouse aRMS primary cell culture showing the combination of imatinib and PP2 to have an additive effect compared to imatinib or PP2 alone. (B) Western blot analysis of an imatinib-resistant mouse aRMS primary culture lysates showing a decrease in the levels of phospho-Mapk (p-Mapk) and phospho-BRaf (p-BRaf) upon treatment with increasing amounts of PP2. (C) Cell viability assay for an imatinib-resistant aRMS primary cell culture revealing that the combination of sorafenib and PP2 has no additive effect on inhibiting cell growth. (D) In vitro cell growth assay for a Pdgfra knock-out mouse rhabdomyosarcoma primary cell culture showing that PP2 had no effect on cell viability. (E) Western blot analysis of a Pdgfra knockout mouse aRMS primary cell culture showing absence of Pdgfra but ongoing activation of BRaf.

Figure 3. Sorafenib efficacy for aRMS

(A) Cell viability assay showing sorafenib being equally effective in inhibiting the growth of naïve (not pretreated) or imatinib-resistant mouse rhabdomyosarcoma primary cell cultures. Sorafenib sensitivity was also similar for embryonal rhabdomyosarcoma (eRMS) and aRMS across species (Fig. S1B). (B-C) Kaplan-Meier survival curves showing sorafenib treatment prolonging event-free survival (EFS) in mice with tumors which measured (B) less than 0.25 cc at diagnosis and (C) larger than 0.25 cc at diagnosis. For EFS, the events were defined as tumor size growing >0.25 cc and >0.75 cc respectively. Tumor growth curves for Fig. 3B and 3C are presented as supplementary figures S2A–B.

Figure 4. Model of the Pdgfra/Src/Raf/Mapk axis in rhabdomyosarcoma

A model depicting the Pdgfra/Src/Raf/MAPK signaling axis in aRMS cells which express Pdgfra and in cells which do not express Pdgfra. We speculate that an RTK other than PDGFRA may activate Src or BRAF under conditions of evolving PDGFRA resistance. In the absence of PDGFRA, Raf signaling remains vital to tumor cell survival.