Targeting a broad spectrum of KRAS-mutant cancers by hyperactivation-induced cell death

Author's response

Johanna Lilja shared

Thank you for the stimulating discussion about our findings.

1) Your findings suggest that SHANK3 expression may influence tumor growth in KRAS-mutant cancers. Is higher expression of SHANK3 associated with tumor size/grades or other clinical parameters in patient cohorts?

This is an interesting and very relevant question. We are currently investigating SHANK3 protein levels and its tissue distribution in human tumors harboring wild-type or mutant KRAS. Notably, SHANK3 scaffold protein exists in numerous isoforms, and these may carry out divergent biological functions at different subcellular localizations. Therefore, it is especially important to investigate the expression of the long SHANK3 isoform (containing N-terminal SPN domain) and its association with clinical parameters in patient cohorts.

Interestingly, SHANK3 mRNA levels are abundant in normal epithelial tissues compared to primary lung and pancreatic tumors, two cancer types, which have a characteristically high frequency of KRAS mutations (http://firebrowse.org). This supports our findings that tumors dependent on oncogenic KRAS-MAPK signaling require low – optimal level of SHANK3 to sustain tumorigenic growth. In KRAS-mutant tumors, SHANK3 fine-tunes the RAS-MAPK signaling to levels supportive of tumor growth and yet below the toxic level that triggers apoptosis.

2) How is the expression of SHANK3 regulated in the cell? Could active KRAS be reciprocally regulating SHANK3 expression/activity?

This is an interesting question that remains to be investigated – especially in cancer cells. As SHANK3 is an abundant core synaptic protein, previous studies have investigated the regulation on SHANK3 expression in the central nervous system (CNS). Shank3 gene expression, and Shank3 protein stability are tightly controlled by multiple mechanisms from the transcriptional to post-translational levels. DNA methylation controls tissue-specific Shank3 expression [1-3], and miRNAs regulate the post-transcriptional Shank3 expression in neurons [4,5]. Shank3 is also heavily ubiquitinated and its protein levels are regulated by the ubiquitin-dependent proteasomal degradation in synapses [6,7]. Notably, there are several Shank3 isoforms due to intragenic promoters and alternative splicing and these isoform have brain region- and development-specific expression patterns [8].

Interestingly, ERK2 was shown to associate with and phosphorylate Shank3 to increase Shank3 turnover by inducing its ubiquitination and degradation in human medulloblastoma-derived cells and neurons [7]. Accordingly, the inhibition of MAPK/ERK pathway by MEK inhibitors or by ERK2 depletion showed, as expected, decreased ERK activation but interestingly, also increased Shank3 protein abundance in neurons. In addition, several other kinases in the MAPK/ERK-pathway, such as KIT, IGF1R, PKA, RAF1 or MEK1, destabilized Shank3 [7]. In line with these findings, we have also observed that MEK inhibition augments SHANK3 protein expression in KRAS-mutant cancer cells. This suggests that SHANK3 levels are regulated by a feedback loop that downregulates SHANK3 protein levels to sustain optimal RAS-ERK signaling. However, future studies are required to fully understand the regulation of SHANK3 levels in cancer cells.

3) You have generated some nanobodies that can disrupt SHANK3-KRAS interaction to induce apoptosis in cancer cells. Do you have plans to push these inhibitors for pharmaceutical development and how will you achieve this? Are there any specific challenges?

As an initial proof-of-concept approach of the feasibility of SHANK3-based therapy, we have generated nanobodies that interfere with the KRAS-SHANK3 interaction. These induce cell death in KRAS-mutant cancer cells in vitro and in vivo, indicating that disruption of the KRAS-SHANK3 interaction could be a viable therapeutic strategy. Development of small-molecule protein-protein interaction inhibitors can be challenging. However, molecules of this class have been developed for other targets and we are open to exploring this possibility also as an option for SHANK3-based therapies.

4) Lower expression of Shank3 mRNA in specific brain regions is associated with autism, both in rodents and humans (PMID: 22749736). Did you consider this issue in your preprint?

SHANK3 is indeed a multifunctional protein with key roles in the CNS. Missense mutations in the SPN domain of SHANK3 have been detected in patients with autism, highlighting the importance of the SPN domain for proper function of SHANK3 [9,10]. We have previously shown that these autism-related mutations within the SHANK3 SPN domain (R12C and L68P) disrupt G-protein interaction and fail to counteract integrin activation along Rap1-RIAM-talin axis in neurons [10]. Furthermore, we have demonstrated that SHANK3 interaction with actin is an important regulator of dendritic spine maturation and normal neuronal function in zebra fish embryos [11]. However, most of the studies on SHANK3 in the CNS involve SHANK3 disruption already during development or long-term depletion of the protein in the adult. Shorter-term inhibition of a specific domain/isoform of SHANK3 in cancer might have limited CNS effects. Nevertheless, this is something that indeed needs to be taking into consideration when designing modalities disrupting SHANK3-KRAS interaction in cancer cells. The blood–brain barrier (BBB) provides protection for neuronal tissues and limits the brain uptake of most pharmaceuticals, which is usually considered as a disadvantage [12]. However, non-BBB permeable anti-SHANK3 drugs would minimize possible off-target effects and adverse side effects in brain tissues.

References:

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