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

Johanna Lilja, Jasmin Kaivola, James R.W. Conway, Joni Vuorio, Hanna Parkkola, Pekka Roivas, Taru Varila, Guillaume Jacquemet, Emilia Peuhu, Emily Wang, Ulla Pentikäinen, Itziar Martinez D. Posada, Hellyeh Hamidi, Arafat K. Najumudeen, Owen J. Sansom, Igor L. Barsukov, Daniel Abankwa, Ilpo Vattulainen, Marko Salmi, Johanna Ivaska

Preprint posted on 21 September 2022 https://www.biorxiv.org/content/10.1101/2022.09.21.508660v1

About one-fifth of all human cancers are driven by KRAS mutations, so can KRAS mutations be targeted to combat cancer? Lilja et al. propose a novel strategy to counteract oncogenic KRAS mutations by disrupting interaction between KRAS and SHANK3

Selected by Yvonne Xinyi Lim

Background:

About 20% of all solid tumors have activating mutations in the KRAS gene. This is especially so in cancers that are often fatal and difficult to treat, such as colorectal, pancreatic, and non-small cell lung cancers1. Once deemed as an undruggable oncoprotein, it is now possible to target active KRAS through inhibitors such as sotorasib and adagrasib. However, these drugs are only effective in a certain subset of patients with KRASG12C mutation, while for those with other KRAS mutations there is still no viable targeted treatment option available 1. Therefore, alternative solutions to target cancers driven by pan-KRAS mutations are direly needed.

In this preprint, the authors present the multidomain scaffold protein SH3 and multiple ankyrin repeat domain 3 (SHANK3) as a novel therapeutic target for pan-KRAS mutant cancers. SHANK3 was found to directly interact with active KRAS to block Ras-MAPK signaling. Disruption of SHANK3 through RNA interference and pharmacological targeting limited the hyperactivation of oncogenic KRAS signaling and inhibited survival and growth of KRAS-mutant cancers. Overall, the findings in this preprint provide valuable insights into the mechanistic regulation of KRAS and suggest potential strategies to counteract mutant KRAS-dependent cancers.

Key findings:

  1. Depletion of SHANK3 impaired cell proliferation in a panel of KRAS-mutant cancer cell lines

To investigate the functional role of SHANK3, the authors used two SHANK3-targeting siRNAs on a panel of twelve human pancreatic, colon and lung cancer cell lines. Cell proliferation and colony growth were significantly impaired in SHANK3-knockdown cell lines with known KRAS mutations (G12D, G12V and G12C), but not in those with wild-type KRAS. This result was reproducible in an in vivo model using the chick embryo chorioallantoic membrane (CAM). This suggests that SHANK3 promotes cell growth only in cancer cell lines driven by activating KRAS mutations.

  1. SHANK3 interacts with active, but not wild-type KRAS

SHANK3 was previously reported to contain an N-terminal SPN domain that adopts a RAS-association domain-like structure and has a high affinity for GTP-bound Ras2. Thus, the authors hypothesized that SHANK3 may bind to active KRAS. To test this hypothesis, multimodal approaches were adopted. Microscale thermophoresis and isothermal titration calorimetry experiments revealed that the purified peptide containing the SPN domain of SHANK3 binds to all active KRAS mutants with similar affinities. However, no interaction with GDP-bound (inactive) KRAS was detected. Therefore, SHANK3 is likely to associate with active KRAS through its SPN domain. To verify this interaction in cells, the authors performed in vitro pulldown and Förster Resonance Energy Transfer (FRET) assays. Their results confirmed that the SHANK3 SPN domain indeed interacts with active KRAS in KRAS-mutant cells. The R12 and K22 residues of the SHANK3 SPN domain are critical in mediating this interaction. Furthermore, growth suppression mediated by SHANK3 silencing could only be rescued by the overexpression of wild-type SHANK3, but not a KRAS-interaction defective SHANK3 mutant. In summary, the interaction between SHANK3 and KRAS is critical for mutant KRAS-driven cell growth and survival.

  1. SHANK3 competes with RAF for KRAS binding to inhibit downstream MAPK signaling and tumor growth

As RAF is a known binding partner of KRAS, the authors proceeded to investigate if SHANK3 can disrupt the KRAS- RAF association. This hypothesis was strongly supported by simulation modelling and in vitro competition binding assays. Elevated SHANK3 SPN domain levels led to a reduction of RAF binding to KRAS. Consequently, downstream ERK1/2 signaling was also diminished in KRAS-mutant cells overexpressing SHANK3 SPN. Further experiments also showed that SHANK3 depletion induced KRAS-mutant cell apoptosis in an ERK-dependent manner. Together, these data demonstrate that the SHANK3 domain of SPN competes with RAF for active KRAS binding and represses the RAS-MAPK pathway. This in turn promotes apoptosis and inhibits growth in KRAS-mutant cells both in vitro and in vivo.

  1. SHANK3 can be pharmacologically targeted to drive KRAS-mutant cells into apoptosis

To move toward the pharmacological development of a SHANK3-targeted therapy, the authors generated nanobodies using a phage display library screen. The nanobodies robustly inhibited SHANK3-KRAS interaction and induced apoptosis in KRAS-mutant cell lines. KRAS-driven tumor growth was also reduced in CAM xenograft models by the use of SHANK3-targeting nanobodies.

What I like about this preprint:

Scientists have known for decades that genomic KRAS alterations have debilitating effects that lead to cancer However, drugs blocking active KRAS were only granted approval  to be used in the clinic in the last two years. This preprint is very timely because the limitations of present KRAS inhibitors are now being uncovered. Current KRAS-targeted drug options are only effective in patients with G12C mutation and significant side effects have been observed even in this patient subset. The authors have provided a neat alternative by identifying a binding partner, SHANK3, that can modulate all active forms of KRAS. The findings of this preprint are promising for to the following reasons: (1) Effects of SHANK3 manipulation are significantly different in KRAS-mutant cancer cell lines, as compared to cell lines with wild-type KRAS. This makes SHANK3 a highly attractive target for KRAS-mutant cancers. (2) The authors used multiple approaches to validate the interaction between KRAS and SHANK3 with high reproducibility between assays. (3) The biological and clinical implications of manipulating the KRAS-SHANK3 interaction is well explained in their experimental data and manuscript writing.

Questions for the authors:

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

References

1          Punekar, S. R., Velcheti, V., Neel, B. G. & Wong, K.-K. The current state of the art and future trends in RAS-targeted cancer therapies. Nature Reviews Clinical Oncology 19, 637-655, doi:10.1038/s41571-022-00671-9 (2022).

2          Lilja, J. et al. SHANK proteins limit integrin activation by directly interacting with Rap1 and R-Ras. Nature Cell Biology 19, 292-305, doi:10.1038/ncb3487 (2017).

 

 

Posted on: 23 October 2022

doi: https://doi.org/10.1242/prelights.32975

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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:

  1. Ching TT, Maunakea AK, Jun P, Hong C, Zardo G, Pinkel D, et al. Epigenome analyses using BAC microarrays identify evolutionary conservation of tissue-specific methylation of SHANK3. Nat Genet. 2005;37(6):645–51. doi:10.1038/ng1563
  2. Maunakea AK, Nagarajan RP, Bilenky M, Ballinger TJ, D’Souza C, Fouse SD, et al. Conserved role of intragenic DNA methylation in regulating alternative promoters. Nature. 2010;466(7303):253–7. doi:10.1038/nature09165.
  3. Beri S, Tonna N, Menozzi G, Bonaglia MC, Sala C, Giorda R. DNA methylation regulates tissue-specific expression of Shank3. J Neurochem. 2007 Jun;101(5):1380-91. doi: 10.1111/j.1471-4159.2007.04539.x. Epub 2007 Apr 10. PMID: 17419801.Kerrisk Campbell M, Sheng M. USP8 Deubiquitinates SHANK3 to Control Synapse Density and SHANK3 Activity-Dependent Protein Levels. J Neurosci. 2018 Jun 6;38(23):5289-5301. doi: 10.1523/JNEUROSCI.3305-17.2018. Epub 2018 May 7. PMID: 29735556; PMCID: PMC6596000.
  4. Choi SY, Pang K, Kim JY, Ryu JR, Kang H, Liu Z, Kim WK, Sun W, Kim H, Han K. Post-transcriptional regulation of SHANK3 expression by microRNAs related to multiple neuropsychiatric disorders. Mol Brain. 2015 Nov 16;8(1):74. doi: 10.1186/s13041-015-0165-3. PMID: 26572867; PMCID: PMC4647645.
  5. Lu J, Zhu Y, Williams S, Watts M, Tonta MA, Coleman HA, Parkington HC, Claudianos C. Autism-associated miR-873 regulates ARID1B, SHANK3 and NRXN2 involved in neurodevelopment. Transl Psychiatry. 2020 Dec 1;10(1):418. doi: 10.1038/s41398-020-01106-8. PMID: 33262327; PMCID: PMC7708977.
  6. Kerrisk Campbell M, Sheng M. USP8 Deubiquitinates SHANK3 to Control Synapse Density and SHANK3 Activity-Dependent Protein Levels. J Neurosci. 2018 Jun 6;38(23):5289-5301. doi: 10.1523/JNEUROSCI.3305-17.2018. Epub 2018 May 7. PMID: 29735556; PMCID: PMC6596000.
  7. Wang L, Adamski CJ, Bondar VV, Craigen E, Collette JR, Pang K, Han K, Jain A, Y Jung S, Liu Z, Sifers RN, Holder JL Jr, Zoghbi HY. A kinome-wide RNAi screen identifies ERK2 as a druggable regulator of Shank3 stability. Mol Psychiatry. 2020 Oct;25(10):2504-2516. doi: 10.1038/s41380-018-0325-9. Epub 2019 Jan 29. PMID: 30696942; PMCID: PMC6663662.
  8. Wang X, Xu Q, Bey AL, Lee Y, Jiang YH. Transcriptional and functional complexity of Shank3 provides a molecular framework to understand the phenotypic heterogeneity of SHANK3 causing autism and Shank3 mutant mice. Mol Autism. 2014 Apr 25;5:30. doi: 10.1186/2040-2392-5-30. PMID: 25071925; PMCID: PMC4113141.
  9. Hassani Nia F, Kreienkamp HJ. Functional Relevance of Missense Mutations Affecting the N-Terminal Part of Shank3 Found in Autistic Patients. Front Mol Neurosci. 2018 Aug 7;11:268. doi: 10.3389/fnmol.2018.00268. PMID: 30131675; PMCID: PMC6090658.
  10. Lilja J, Zacharchenko T, Georgiadou M, Jacquemet G, De Franceschi N, Peuhu E, Hamidi H, Pouwels J, Martens V, Nia FH, Beifuss M, Boeckers T, Kreienkamp HJ, Barsukov IL, Ivaska J. SHANK proteins limit integrin activation by directly interacting with Rap1 and R-Ras. Nat Cell Biol. 2017 Apr;19(4):292-305. doi: 10.1038/ncb3487. Epub 2017 Mar 6. PMID: 28263956; PMCID: PMC5386136.
  11. Salomaa SI, Miihkinen M, Kremneva E, Paatero I, Lilja J, Jacquemet G, Vuorio J, Antenucci L, Kogan K, Hassani Nia F, Hollos P, Isomursu A, Vattulainen I, Coffey ET, Kreienkamp HJ, Lappalainen P, Ivaska J. SHANK3 conformation regulates direct actin binding and crosstalk with Rap1 signaling. Curr Biol. 2021 Nov 22;31(22):4956-4970.e9. doi: 10.1016/j.cub.2021.09.022. Epub 2021 Oct 4. PMID: 34610274.
  12. Arvanitis CD, Ferraro GB, Jain RK. The blood-brain barrier and blood-tumour barrier in brain tumours and metastases. Nat Rev Cancer. 2020 Jan;20(1):26-41. doi: 10.1038/s41568-019-0205-x. Epub 2019 Oct 10. PMID: 31601988; PMCID: PMC8246629.

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