CDK4/6 Inhibition Induces Senescence and Enhances Radiation Response by Disabling DNA Damage Repair in Oral Cavity Squamous Cell Carcinoma.

PURPOSE: HPV(-) OCSCC resists radiation treatment. The CDKN2A gene, encoding p16INK4A, is commonly disrupted in OCSCC. p16 inhibits CDK4/CDK6, leading to cell cycle arrest, but the biological sequelae of CDK4/6 inhibition in OCSCC remains understudied. This study examines whether inhibition of CDK4/6 enhances radiation response in OCSCC. METHODS: MTT assays were performed in OCSCC cell lines HN5 and CAL27 following treatment with palbociclib. Clonogenic survival and synergy were analyzed after radiation (RT-2 or 4Gy), palbociclib (P) (0.5 µM or 1 µM), or concurrent combination treatment (P+RT). DNA damage/repair and senescence were examined. CDK4/6 were targeted via siRNA to corroborate P+RT effects. Three-dimensional immortalized spheroids and organoids derived from patient tumors (conditionally reprogrammed OCSCC CR-06 and CR-18) were established to further examine and validate responses to P+RT. RESULTS: P+RT demonstrated reduced viability and synergy, increased ß-gal expression (~95%), and ~two-fold higher γH2AX. Rad51 and Ku80 were reduced after P+RT, indicating impairment of both HR and NHEJ. siCDK4/6 increased senescence with radiation. Spheroids showed reduced proliferation and size with P+RT. CR-06 and CR-18 further demonstrated three-fold reduced proliferation and organoids size with P+RT. CONCLUSION: Targeting CDK4/6 can lead to improved efficacy when combined with radiation in OCSCC by inducing senescence and inhibiting DNA damage repair.

Palbociclib Renders Human Papilloma Virus-Negative Head and Neck Squamous Cell Carcinoma Vulnerable to the Senolytic Agent Navitoclax.

We demonstrate that inhibition of cyclin-dependent kinases 4/6 (CDK4/6) leads to senescence in human papillomavirus (HPV)-negative (-) head and neck squamous cell carcinoma (HNSCC), but not in HPV-positive (+) HNSCC. The BCL-2 family inhibitor, navitoclax, has been shown to eliminate senescent cells effectively. We evaluated the efficacy of combining palbociclib and navitoclax in HPV- HNSCC. Three HPV- HNSCC cell lines (CAL27, HN31, and PCI15B) and three HPV+ HNSCC cell lines (UPCI-SCC-090, UPCI-SCC-154, and UM-SCC-47) were treated with palbociclib. Treatment drove reduced expression of phosphorylated Rb (p-Rb) and phenotypic evidence of senescence in all HPV- cell lines, whereas HPV+ cell lines did not display a consistent response by Rb or p-Rb and did not exhibit morphologic changes of senescence in response to palbociclib. In addition, treatment of HPV- cells with palbociclib increased both ß-galactosidase protein expression and BCL-xL protein expression compared with untreated controls in HPV- cells. Co-expression of ß-galactosidase and BCL-xL occurred consistently, indicating elevated BCL-xL expression in senescent cells. Combining palbociclib with navitoclax led to decreased HPV- HNSCC cell survival and led to increased apoptosis levels in HPV- cell lines compared with each agent given alone. IMPLICATIONS: This work exploits a key genomic hallmark of HPV- HNSCC (CDKN2A disruption) using palbociclib to induce BCL-xL-dependent senescence, which subsequently causes the cancer cells to be vulnerable to the senolytic agent, navitoclax.

Apoptosis signaling molecules as treatment targets in head and neck squamous cell carcinoma.

OBJECTIVES: To evaluate BCL-2 family signaling molecules in head and neck squamous cell carcinoma (HNSCC) and examine the ability of therapeutic agents with variable mechanisms of action to induce apoptosis in HNSCC cells. METHODS: messenger ribonculeic acid (mRNA) expression of BAK, BAX, B-cell lymphoma (Bcl-2), BCL2 Like 1 (BCL2L1), and MCL1 were measured in The Cancer Genome Atlas (TCGA) head and neck cancer dataset, as well as in a dataset from a cohort at Montefiore Medical Center (MMC). Protein expression was similarly evaluated in a panel of HNSCC cell lines (HN30, HN31, HN5, MDA686LN, UMSCC47). Cell viability and Annexin V assays were used to assess the efficacy and apoptotic potential of a variety of agents (ABT-263 [navitoclax], A-1210477, and bortezomib. RESULTS: Expression of BAK, BAX, BCL2L1, and MCL1 were each significantly higher than expression of BCL2 in the TCGA and MMC datasets. Protein expression demonstrated the same pattern of expression when examined in HNSCC cell lines. Treatment with combined ABT-263 (navitoclax)/A-1210477 or with bortezomib demonstrated apoptosis responses that approached or exceeded treatment with staurospaurine control. CONCLUSION: HNSCC cells rely on inhibition of apoptosis via BCL-xL and MCL-1 overexpression, and induction of apoptosis remains a potential therapeutic option as long as strategies overcome redundant anti-apoptotic signals. LEVEL OF EVIDENCE: NA Laryngoscope, 130:2643-2649, 2020.

Optimal targeting of BCL-family proteins in head and neck squamous cell carcinoma requires inhibition of both BCL-xL and MCL-1.

Mechanisms of treatment resistance in head and neck squamous cell carcinoma (HNSCC) are not well characterized. In this study, HNSCC tumors from a cohort of prospectively enrolled subjects on an ongoing tissue banking study were divided into those that persisted or recurred locoregionally (n=23) and those that responded without recurrence (n=35). Gene expression was evaluated using llumina HumanHT-12-v3 Expression BeadChip microarrays. Sparse Partial Least Squares - Discriminant Analysis (sPLS-DA) identified 135 genes discriminating treatment-resistant from treatment-sensitive tumors. BCL-xL was identified among 23% of canonical pathways derived from this set of genes using Ingenuity Pathway analysis. The BCL-xL protein was expressed in 8 HNSCC cell lines examined. Cells were treated with the BCL-xL inhibitor, ABT-263 (navitoclax): the average half maximal inhibitory concentration (IC50) was 8.9µM (range 6.6µM - 13.9µM). Combining ABT-263 did not significantly increase responses to 2 Gy radiation or cisplatin in the majority of cell lines. MCL-1, a potential mediator of resistance to ABT-263, was expressed in all cell lines and HNSCC patient tumors, in addition to BCL-xL. Treatment with the MCL-1 inhibitor, A-1210477, in HNSCC cell lines showed an average IC50 of 10.7µM (range, 8.8µM to 12.7µM). Adding A-1210477 to ABT-263 (navitoclax) treatment resulted in an average 7-fold reduction in the required lethal dose of ABT-263 (navitoclax) when measured across all 8 cell lines. Synergistic activity was confirmed in PCI15B, Detroit 562, MDA686LN, and HN30 based on Bliss Independence analysis. This study demonstrates that targeting both BCL-xL and MCL-1 is required to optimally inhibit BCL-family pro-survival molecules in HNSCC, and co-inhibition is synergistic in HNSCC cancer cells.

AHNS Series: Do you know your guidelines? Principles of treatment for locally advanced or unresectable head and neck squamous cell carcinoma.

This article is a continuation of the "Do You Know Your Guidelines" series, initiated by the Education committee of the American Head and Neck Society. Treatment guidelines for advanced head and neck squamous cell carcinoma are reviewed here, including the critical roles of radiotherapy, chemotherapy, and the recent application of immunotherapy agents. We will be limiting this discussion to include cancers of the oral cavity, oropharynx, hypopharynx, and larynx. It should be noted that much of the article pertains to human papillomavirus (HPV)-negative oropharyngeal cancer where applicable, as HPV-positive oropharyngeal squamous cell carcinoma carries a different natural history, different prognosis, and now different staging criteria. Additionally, the article will not include information on nasopharyngeal or sinus cancers, as these latter topics are covered in separate "Do you know your guidelines?" installments and these diagnoses carry somewhat different approaches to diagnosis and management that diverge from the focus of this article.