Cancer stem cells (CSCs) play a critical role in the initiation, progression and therapy relapse of many cancers including non-small cell lung cancer (NSCLC). Here, we aimed to address the question of whether the FACS-sorted CSC-like (CD44 + &CD133 +) vs. non-CSC (CD44−/CD133− isogenic subpopulations of p53wt A549 and p53null H1299 cells differ in terms of DNA-damage signaling and the appearance of “dormant” features, including polyploidy, which are early markers (predictors) of their sensitivity to genotoxic stress. X-ray irradiation (IR) at 5 Gy provoked significantly higher levels of the ATR-Chk1/Chk2-pathway activity in CD44−/CD133− and CD133+ subpopulations of H1299 cells compared to the respective subpopulations of A549 cells, which only excited ATR-Chk2 activation as demonstrated by the Multiplex DNA-Damage/Genotoxicity profiling. The CD44+ subpopulations did not demonstrate IR-induced activation of ATR, while significantly augmenting only Chk2 and Chk1/2 in the A549- and H1299-derived cells, respectively. Compared to the A549 cells, all the subpopulations of H1299 cells established an increased IR-induced expression of the γH2AX DNA-repair protein. The CD44−/CD133− and CD133+ subpopulations of the A549 cells revealed IR-induced activation of ATR-p53-p21 cell dormancy signaling-mediated pathway, while none of the CD44+ subpopulations of either cell line possessed any signs of such activity. Our data indicated, for the first time, the transcription factor MITF–FAM3C axis operative in p53-deficient H1299 cells, specifically their CD44+ and CD133+ populations, in response to IR, which warrants further investigation. The p21-mediated quiescence is likely the predominant surviving pathway in CD44−/CD133− and CD133+ populations of A549 cells as indicated by single-cell high-content imaging and analysis of Ki67- and EdU-coupled fluorescence after IR stress. SA-beta-galhistology revealed that cellular-stress-induced premature senescence (SIPS) likely has a significant influence on the temporary dormant state of H1299 cells. For the first time, we demonstrated polyploid giant and/or multinucleated cancer-cell (PGCC/MGCC) fractions mainly featuring the progressively augmenting Ki67low phenotype in CD44+ and CD133+ A549 cells at 24–48 h after IR. In contrast, the Ki67high phenotype enrichment in the same fractions of all the sorted H1299 cells suggested an increase in their cycling/heterochromatin reorganization activity after IR stress. Our results proposed that entering the “quiescence” state rather than p21-mediated SIPS may play a significant role in the survival of p53wt CSC-like NSCLC cells after IR. The results obtained are important for the selection of therapeutic schemes for the treatment of patients with NSCLC, depending on the functioning of the p53 system in tumor cells.

https://www.mdpi.com/1422-0067/23/9/4922/pdf?version=1651808911

CD44+ and CD133+ Non-Small Cell Lung Cancer Cells Exhibit DNA Damage Response Pathways and Dormant Polyploid Giant Cancer Cell Enrichment Relating to Their p53 Status

Abstract Mutational loss of CDKN2A (encoding p16INK4A) tumor-suppressor function is a key genetic step that complements activation of KRAS in promoting the development and malignant growth of pancreatic ductal adenocarcinoma (PDAC). However, pharmacologic restoration of p16INK4A function with inhibitors of CDK4 and CDK6 (CDK4/6) has shown limited clinical efficacy in PDAC. Here, we found that concurrent treatment with both a CDK4/6 inhibitor (CDK4/6i) and an ERK–MAPK inhibitor (ERKi) synergistically suppresses the growth of PDAC cell lines and organoids by cooperatively blocking CDK4/6i-induced compensatory upregulation of ERK, PI3K, antiapoptotic signaling, and MYC expression. On the basis of these findings, a Phase I clinical trial was initiated to evaluate the ERKi ulixertinib in combination with the CDK4/6i palbociclib in patients with advanced PDAC (NCT03454035). As inhibition of other proteins might also counter CDK4/6i-mediated signaling changes to increase cellular CDK4/6i sensitivity, a CRISPR-Cas9 loss-of-function screen was conducted that revealed a spectrum of functionally diverse genes whose loss enhanced CDK4/6i growth inhibitory activity. These genes were enriched around diverse signaling nodes, including cell-cycle regulatory proteins centered on CDK2 activation, PI3K–AKT–mTOR signaling, SRC family kinases, HDAC proteins, autophagy-activating pathways, chromosome regulation and maintenance, and DNA damage and repair pathways. Novel therapeutic combinations were validated using siRNA and small-molecule inhibitor–based approaches. In addition, genes whose loss imparts a survival advantage were identified (e.g., RB1, PTEN, FBXW7), suggesting possible resistance mechanisms to CDK4/6 inhibition. In summary, this study has identified novel combinations with CDK4/6i that may have clinical benefit to patients with PDAC. Significance: CRISPR-Cas9 screening and protein activity mapping reveal combinations that increase potency of CDK4/6 inhibitors and overcome drug-induced compensations in pancreatic cancer. 

Combination Therapies with CDK4/6 Inhibitors to Treat <i>KRAS-</i>Mutant Pancreatic Cancer

Pancreatic cancer is highly aggressive and lethal. Due to the lack of effective methods for detecting the disease at an early stage, pancreatic cancer is frequently diagnosed late. Gemcitabine has been the standard chemotherapy drug for patients with pancreatic cancer for over 20 years, but its anti-tumor effect is limited. Therefore, FOLFIRINOX (leucovorin, fluorouracil, irinotecan, oxaliplatin) as well as combination therapies using gemcitabine and conventional agents, such as cisplatin and capecitabine, has also been administered; however, these have not resulted in complete remission. Therefore, there is a need to develop novel and effective therapies for pancreatic cancer. Recently, some studies have reported that combinations of gemcitabine and targeted drugs have had significant anti-tumor effects on pancreatic cancer cells. As gemcitabine induced DNA damage response, the proteins related to DNA damage response can be suitable additional targets for novel gemcitabine-based combination therapy. Furthermore, KRAS/ RAF/MEK/ERK signaling triggered by oncogenic mutated KRAS and autophagy are frequently activated in pancreatic cancer. Therefore, these characteristics of pancreatic cancer are potential targets for developing effective novel therapies. In this minireview, combinations of gemcitabine and targeted drugs to these characteristics, combinations of targeted drugs, combinations of natural products and anti-cancer agents, including gemcitabine, and combinations among natural products are discussed.

Effective combinations of anti-cancer and targeted drugs for pancreatic cancer treatment

Molecular docking/dynamic simulations, MEP, ADME-TOX-based analysis of xanthone derivatives as CHK1 inhibitors

Aneuploidy is a hallmark of human cancer, yet the cellular mechanisms that allow cells to cope with aneuploidy-induced cellular stresses remain largely unknown. Such coping mechanisms may present cellular vulnerabilities that can be harnessed for targeting cancer cells. Here, we induced aneuploidy in non-transformed RPE1-hTERT cells and derived multiple stable clones with various degrees of chromosome imbalances. We performed an unbiased genomic profiling of 6 isogenic clones, using whole-exome and RNA sequencing. We then functionally interrogated their cellular dependency landscapes, using genome-wide CRISPR/Cas9 screens and large-scale drug screens. We found that aneuploid clones activated the DNA damage response (DDR), and were consequently more resistant to further DNA damage induction. Interestingly, aneuploid cells also exhibited elevated RAF/MEK/ERK pathway activity, and were more sensitive to several clinically-relevant drugs targeting this pathway, and in particular to genetic and chemical CRAF inhibition. CRAF activity was functionally linked to the resistance to DNA damage induction, as CRAF inhibition sensitized aneuploid cells to DNA damage-inducing chemotherapies. The association between aneuploidy, RAF/MEK/ERK signaling, and DDR was independent of p53. The increased activity and dependency of aneuploid cells on the RAF/MEK/ERK pathway was validated in another isogenic aneuploid system, and across hundreds of human cancer cell lines, confirming their relevance to human cancer. Overall, our study provides a comprehensive resource for genetically-matched karyotypically-stable cells of various aneuploidy states, and reveals a novel therapeutically-relevant cellular dependency of aneuploid cells.

/pdf/human-aneuploid-cells-depend-on-the-raf-mek-erk-pathway-for-2gipt24q.pdf

Kristina Drizyte-Miller

Papers

CHK1 protects oncogenic KRAS-expressing cells from DNA damage and is a target for pancreatic cancer treatment.