CDK4/6 inhibition protects normal epithelia from chemotherapy induced gut toxicity

Introduction

Chemotherapy relies on a high ratio of toxicity toward cancer cells vs. non-malignant cells. Normal tissues with a high proportion of proliferating cells such as the epithelia of the gut, are most vulnerable toward drug induced toxicity.

The destruction of gut epithelia can give rise to dose-limiting and life-threatening side effects of chemotherapeutics, and it is therefore of high importance to evaluate strategies for avoiding intestinal toxicities during cancer therapy.

In this paper, the Author’s use a murine intestinal organoid model to assess a strategy for protecting the gut mucosa against chemotherapy, taking advantage of the cell cycle specificity of many cancer drugs.

Main Points

• To establish whether the organoids were sensitive to cyclin-dependent kinase inhibition, organoids were treated for 48 hours with the CDK4 and CDK6 inhibitor Palbociclib with a subsequent washout. Interestingly, although this transient treatment regimen moderately slowed down the growth of the organoids, it did not induce any prolonged toxicity on the gut epithelia.
  Furthermore, the organoids did not display any morphological signs of increased damage and resumed proliferation and showed similar growth kinetics to control after the treatment was removed. Organoid growth was quantified by measuring the total area of viable organoids per well using bright field images taken daily using a semi-automated cell imager system. The area of organoids on day 1 was set 100%, and the organoid growth was normalized to day 1.
• Immunoblot analysis of Palbociclib treated intestinal organoids showed reduced phosphorylation of the retinoblastoma protein (pRb) and E2F-mediated transcription. Furthermore, immunohistochemistry showed reduced levels of Ki-67 expression indicated a decreased cell proliferation.
• CDK4/6 inhibitors represent clinically established oncotherapeutics, and mainly arrests cells in G1 phase, reducing the proportion of cells that go through S- and M-phases. CDK4/6 inhibition induces pRb hypo-phosphorylation, which mediates cell cycle arrest.
• To investigate if CDK4/6 inhibition could protect from Gemcitabine induced toxicity, intestinal organoids were pre-treated for 24 hours with Palbociclib before Gemcitabine was added in combination with Palbociclib for another 24 hours. Cell viability assays showed that pre-treatment with Palbociclib significantly increased organoid viability compared to treatment with the S-phase specific DNA synthesis inhibitor (Gemcitabine) alone, which induced significant toxicity of the gut epithelia.
• In contrast, pre-treatment with Palbociclib did not protect the organoids from Cisplatin-induced toxicity, which mediates DNA cross-links by cell cycle-independent mechanisms.
• In addition, pre-treatment of RB1-mutant cancer cells lines with Palbociclib did not protect from chemotherapy induced toxicity.
• Next the Authors explored if Palbociclib could protect gut epithelium against the cytotoxic effects induced by the topoisomerase I inhibitor Irinotecan. Organoids pre-treated with Palbociclib before parallel treatment with SN-38 (the active metabolite of Irinotecan) resulted in increased organoid viability and reduced levels of cleaved caspase-3 and DNA damage response markers (phosphorylated KAP1 and ϒ-H2A×) compared to organoids treated with SN-38 alone. This strongly suggests that CDK4/6 inhibition preserved DNA integrity in the presence of a topoisomerase inhibitor.
  Furthermore, pre-treated organoids resumed their growth 2 days after the removal of SN-38 whilst it took SN-38 only treated organoids at least 5 days to start growing.

Conclusion

In this paper, the Authors show that retreatment with the CDK4/6 inhibitor Palbociclib induces a transient cell cycle arrest in the organoids which protects the cells of organoids against the toxic effects of gemcitabine as well as irinotecan. This raises the perspective of using CDK inhibitors to prevent the intestinal toxicities of chemotherapeutics that act in a cell cycle-specific manner.

EPISTEM SERVICES

Organoids

• If you need to investigate the potential  effect of drug induced toxicity, on proliferating cells such as the epithelia of the gut, Epistem can support you, via our expertise in Organoids.
• Epistem provides 3D organoid models from several species that mimic the intestine, offering valuable insights into biology, disease, and compound effects. These structures allow for multiple assessments, including viability, branching, and gene expression.
• To aid in the assessment we use a Cytation 5 live-cell imaging system.
• If you have project specific requirements, our BD team would be delighted to discuss them with you.
• Epistem has also delivered contract data on CDK inhibitors from various in-house capabilities including IHC, RNA-seq, qPCR and our plucked-hair biomarker platform.