Pyrvinium Pamoate and BCL-XL Inhibitors Act Synergistically to Kill Patient-Derived Colorectal Adenoma Organoids

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

• The Results presented in Table 1 aren't clear: what does «Multiple» mean in the «Number of adenomas» column? If these are sporadic adenomas patients, as stated, and not polyposis patients, how can several of them have «Multiple» adenomas in a colonic segment? And how are these «Multiple» adenomas always of the same »Adenoma type»? Are these really «Multiple» adenomas or simply multiple fragments of the same adenoma?
• The Table 1 Legend is also difficult to understand.

Comments on the Quality of English Language

- Pg 8. Line 250

«and sessile serrated adenomas (n=6) range of sites,» - Text missing?

- Pg 17, Lines 538-39

«Whilst promising results are emerging in the preclinical settings testing combination therapies is difficult but the clinical potential is yet to be shown.» - This sentence needs to be corrected, as do several typos throughout the text.

- Supplementary Figure S2

«Other organoids demonstrate inverted polarity (B). The majority of organoids display E-cadherin at the basolateral surface and apical actin polarityShown are images rendered in Imaris (3D blend mode) (panels A and B) and single confocal slices (A’ slice 30 of 73, A’’ slice 47 of 73, B’ slice 43 of 64, B’’ slice 23 of 64, C’ slice 64of 91, C’’ slice 24 of 91).»

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Sporadic colorectal cancer (CRC) is the most common type of CRC, accounting for 70% to 80% of all cases. It develops without a strong family history or inherited genetic predisposition. Instead, it arises from a combination of environmental, lifestyle, and somatic genetic factors. The evidence that CRC is now affecting a much younger population presents an additional concern. Meta-analyses conducted in patients acquiring CRC at a youthful age have a worse prognosis. Maree C Faux et al. is an intriguing study aimed to determine if targeting the Wnt pathway in combination with pro-apoptotic mimetics altered the proliferative capacity or viability of human colorectal adenoma cells. They used colorectal/cecum organoids, alias colonoids. Patient-derived colorectal adenoma organoid cultures were established from colon adenoma tissue collected by colonoscopy and recapitulated the histopathology of primary colorectal adenoma tissue. The growth of colorectal adenoma organoids is inhibited by the Wnt-signaling antagonist pyrvinium pamoate (PP) and a pro-apoptotic inhibitor of BCL-XL but not by BCL-2 (venetoclax) or MCL-1 inhibitors. At low concentrations the PP and the BCL-XL inhibitor combination demonstrated potent synergy and induced apoptosis in APC defective patient-derived adenoma organoids even in the presence of oncogenic KRAS or BRAF mutations, providing a new strategy for colon cancer prevention. During the cell death analyses of organoids one-way ANOVA test performed with Bonferroni’s correction method for multiple comparisons were appropriate. The results are convincing as supported by Figs. 1 through 5, easy to follow. These results suggest that a new drug combination Wnt antagonist and pro-apoptotic inhibitor could be an effective strategy for killing colon adenomas and preventing CRC. Overall, the findings provide the relevant basis for future approaches to colon cancer prevention and the testing of patient-centered-derived organoids from pre-cancerous stages provides an opportunity for identifying anti-cancer drug combinations likely to improve outcomes for patients.

Obviously, there are limitations to the study:

• The sample size is small.
• There was only one sample with gain of BCL-XL and no Wnt pathway mutation, so interpretation of differences in drug response should be treated cautiously.
• Did not explore the effects of immune components, the involvement of the immune system in adenoma killing is worthy of further investigation.
• The combination treatment resulted in synergistic effects and profound induction of apoptosis compared to individual drug treatments.
• Future work using PDXs from adenoma organoids may be important to measure the toxicity of the drug combinations in an in vivo setting.

Author Response

Please see the attachment

Author Response File: Author Response.pdf