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Supplementary Figure S4 related to Figure 5D-F. (A) Individual tumor volume changes in Detroit 562-xenografted mice treated with vehicle, adavosertib (30 mg/kg, daily, p.o.), alisertib (30 mg/kg, daily, p.o.), or the combinations of alisertib with adavosertib. Tumor volume changes of all mice belonged to this study was shown in bottom panel (n = 6 per group). Significant differences between the combination and single drug alone are shown. (B) Body weight (g) of mice was monitored every other day. Graphs depict Mean {plus minus} SEM.

Supplementary figure legends

Purpose:Human papillomavirus (HPV)-negative head and neck squamous cell carcinomas (HNSCC) commonly bear disruptive mutations in TP53, resulting in treatment resistance. In these patients, direct targeting of p53 has not been successful, but synthetic lethal approaches have promise. Although Aurora A kinase (AURKA) is overexpressed and an oncogenic driver, its inhibition has only modest clinical effects in HPV-negative HNSCC. We explored a novel combination of AURKA and WEE1 inhibition to overcome intrinsic resistance to AURKA inhibition.Experimental Design: AURKA protein expression was determined by fluorescence-based automated quantitative analysis of patient specimens and correlated with survival. We evaluated treatment with the AURKA inhibitor alisertib (MLN8237) and the WEE1 inhibitor adavosertib (AZD1775), alone or in combination, using in vitro and in vivo HNSCC models.Results:Elevated nuclear AURKA correlated with worse survival among patients with p16(−) HNSCC. Alisertib caused spindle defects, G2–M arrest and inhibitory CDK1 phosphorylation, and cytostasis in TP53 mutant HNSCC FaDu and UNC7 cells. Addition of adavosertib to alisertib instead triggered mitotic entry and mitotic catastrophe. Moreover, in FaDu and Detroit 562 xenografts, this combination demonstrated synergistic effects on tumor growth and extended overall survival compared with either vehicle or single-agent treatment.Conclusions:Combinatorial treatment with adavosertib and alisertib leads to synergistic antitumor effects in in vitro and in vivo HNSCC models. These findings suggest a novel rational combination, providing a promising therapeutic avenue for TP53-mutated cancers.

Wound repair requires the coordination of multiple cell types including immune cells and tissue resident cells to coordinate healing and return of tissue function. Diabetic foot ulceration is a type of chronic wound that impacts over 4 million patients in the US and over 7 million worldwide (Edmonds et al., 2021). Yet, the cellular and molecular mechanisms that go awry in these wounds are not fully understood. Here, by profiling chronic foot ulcers from non-diabetic (NDFUs) and diabetic (DFUs) patients using single-cell RNA sequencing, we find that DFUs display transcription changes that implicate reduced keratinocyte differentiation, altered fibroblast function and lineages, and defects in macrophage metabolism, inflammation, and ECM production compared to NDFUs. Furthermore, analysis of cellular interactions reveals major alterations in several signaling pathways that are altered in DFUs. These data provide a view of the mechanisms by which diabetes alters healing of foot ulcers and may provide therapeutic avenues for DFU treatments.

Purpose: Human papillomavirus (HPV)-negative head and neck squamous cell carcinomas (HNSCC) commonly bear disruptive mutations in TP53, resulting in treatment resistance. In these patients, direct targeting of p53 has not been successful, but synthetic lethal approaches have promise. Although Aurora A kinase (AURKA) is overexpressed and an oncogenic driver, its inhibition has only modest clinical effects in HPV-negative HNSCC. We explored a novel combination of AURKA and WEE1 inhibition to overcome intrinsic resistance to AURKA inhibition. Experimental Design: AURKA protein expression was determined by fluorescence-based automated quantitative analysis of patient specimens and correlated with survival. We evaluated treatment with the AURKA inhibitor alisertib (MLN8237) and the WEE1 inhibitor adavosertib (AZD1775), alone or in combination, using in vitro and in vivo HNSCC models. Results: Elevated nuclear AURKA correlated with worse survival among patients with p16(−) HNSCC. Alisertib caused spindle defects, G2–M arrest and inhibitory CDK1 phosphorylation, and cytostasis in TP53 mutant HNSCC FaDu and UNC7 cells. Addition of adavosertib to alisertib instead triggered mitotic entry and mitotic catastrophe. Moreover, in FaDu and Detroit 562 xenografts, this combination demonstrated synergistic effects on tumor growth and extended overall survival compared with either vehicle or single-agent treatment. Conclusions: Combinatorial treatment with adavosertib and alisertib leads to synergistic antitumor effects in in vitro and in vivo HNSCC models. These findings suggest a novel rational combination, providing a promising therapeutic avenue for TP53-mutated cancers.

Resistance to DNA-damaging agents is a significant cause of treatment failure and poor outcomes in oncology. To identify unrecognized regulators of cell survival we performed a whole-genome CRISPR-Cas9 screen using treatment with ionizing radiation as a selective pressure, and identified STING (stimulator of interferon genes) as an intrinsic regulator of tumor cell survival. We show that STING regulates a transcriptional program that controls the generation of reactive oxygen species (ROS), and that STING loss alters ROS homeostasis to reduce DNA damage and to cause therapeutic resistance. In agreement with these data, analysis of tumors from head and neck squamous cell carcinoma patient specimens show that low STING expression is associated with worse outcomes. We also demonstrate that pharmacologic activation of STING enhances the effects of ionizing radiation in vivo, providing a rationale for therapeutic combinations of STING agonists and DNA-damaging agents. These results highlight a role for STING that is beyond its canonical function in cyclic dinucleotide and DNA damage sensing, and identify STING as a regulator of cellular ROS homeostasis and tumor cell susceptibility to reactive oxygen dependent, DNA damaging agents., The endoplasmic reticulum-localized adaptor STING regulates the innate immune response through its ability to sense DNA damage. Here the authors reveal that STING functions as a regulator of cellular ROS homeostasis and tumor cell susceptibility to reactive oxygen dependent, DNA damaging agents.

To understand the diabetic wound healing microenvironment, we profiled 174,962 single cells from foot, forearm, and PBMCs using single-cell RNA sequencing (scRNASeq) approach. Our analysis shows enrichment of a unique population of fibroblasts overexpressing MMP1, MMP3, MMP11, HIF1A, CHI3L1, and TNFAIP6 genes and M1 macrophage polarization in the DFU patients with healing wounds. Further, scRNASeq of spatially separated samples from same patient and spatial transcriptomics (ST) revealed preferential localization of these healing associated fibroblasts toward deep wound/ulcer bed as compared to wound edge or non-wounded skin. ST also validated our findings of higher enrichment of M1 macrophages in healers and M2 macrophages in non-healers. Our analysis provides deep insights into the wound healing microenvironment, identifying cell types that could be critical in promoting DFU healing, and may inform novel therapeutic approaches for DFU treatment.

Introduction Cetuximab, a monoclonal antibody to the epidermal growth factor receptor (EGFR), extends survival in combination with standard therapy in head and neck squamous cell carcinoma (HNSCC). However, as effects are modest, and patients experience side effects, a biomarker to predict resistance and personalize therapy is needed. Activation of signaling pathways downstream from receptor tyrosine kinases predicts resistance to such therapies in other cancers. The most common abnormalities downstream from EGFR in HNSCC are in the PI3K pathway, activated via loss of expression of the regulator PTEN, or via PI3K mutation. We studied whether PTEN and/or PI3K abnormalities predict resistance to cetuximab. Methods Tumor PTEN and PIK3CA/PI3K p110α were analyzed in samples from subjects treated on two trials of cetuximab-based therapy for patients with metastatic or recurrent HNSCC: E5397, a randomized trial of cisplatin plus placebo versus cisplatin plus cetuximab; and NCI-8070, a randomized trial of cetuximab plus sorafenib versus cetuximab. In situ quantification of PTEN and PI3K p110 α was performed using the AQUA™ method of quantitative immunofluorescence. PI3KCA hot spot mutations were determined with BEAMing. Results For E5397, in multivariable analysis, PTEN expressing/PIK3CA WT patients tended to improve PFS with cetuximab compared to placebo (N = 48; HR = 0.54, Wald p = 0.0502). High PTEN expression was significantly associated with superior PFS among patients treated on NCI-8070 (N = 37; HR = 0.35, p = 0.008). Conclusion Loss of PTEN expression may be associated with lack of benefit from cetuximab. This analysis is limited by small sample size, and PTEN as a potential predictive biomarker merits validation in larger sample sets.

The mamillary body (MM) is a group of hypothalamic nuclei related to memory and spatial navigation that interconnects hippocampal, thalamic, and tegmental regions. Here we demonstrate that Lhx5, a LIM-HD domain transcription factor expressed early in the developing posterior hypothalamus, is required for the generation of the MM and its derived tracts. The MM markers Foxb1, Sim2, and Lhx1 are absent in Lhx5 knock-out mice from early embryonic stages, suggesting abnormal specification of this region. This was supported by the absence of Nkx2.1 and expansion of Shh in the prospective mamillary area. Interestingly, we also found an ectopic domain expressing Lhx2 and Lhx9 along the anterio-posterior hypothalamic axis. Our results suggest that Lhx5 controls early aspects of hypothalamic development by regulating gene expression and cellular specification in the prospective MM.

e14105 Background: Aurora Kinase A (AURKA) is overexpressed in HNSCC, and correlates with poor prognosis. It has been identified as a potential therapeutic target, yet the response rate for the AURKA inhibitor MLN8237 is only 9% in treatment refractory HNSCC. We hypothesized that although AURKA inhibitors lead to defective spindle assembly, they may also reduce mitotic entry, undermining cytotoxic effect. We predicted that adding a WEE1 inhibitor to an AURKA inhibitor would mitigate this effect and enhance cell death Methods: Cell viability assays were performed on FaDu ( p53 mut.), Detroit562 ( p53 mut.), and UNC7 ( p53 WT) HPV negative HNSCC cell lines treated with AZD1775 (AZD), MLN8237 (MLN), and combination of AZD+MLN. Oncosphere formation assays were used to confirm findings of cell death, and western blot analysis and confocal microscopy were used to investigate mechanism of synergy. The above drugs were also given at varying doses via oral gavage to FaDu xenografted nude mice. Results: There was clear synergy of AZD and MLN in-vitro. Combination Indices were determined by the Chou-Talalay method: FaDu 0.4, Detroit562 0.5, and UNC7 0.6 (synergy = < 0.8). Oncopshere assays showed inability of AZD+MLN treated cells to re-differentiate. FaDu cells treated with MLN had increased p-CDK1 and reduced phospho-histone H3 (pHH3), suggesting reduced mitotic entry. AZD+MLN treated cells had reduced p-CDK1 and increased pHH3, similar to AZD treated cells; they also had spindle disarray with poor chromatin organization on confocal microscopy indicating mitotic catastrophe. In mice, the combination of AZD+MLN inhibited tumor growth, with no apparent toxicity. Mice treated with either drug alone had tumor volumes over 1000mm3 and were sacrificed at day 21; those treated with AZD 90mg/kg and MLN 30mg/kg had tumors volumes around 300mm3 on day 28. Conclusions: AZD and MLN synergistically enhance cell death in HNSCC cell lines and significantly inhibit tumor growth in mouse xenograft models. The ability of AZD to overcome intrinsic resistance to MLN may underlie mechanism of synergy. We recommend further investigation of AURKA and WEE1 inhibition in other cancers with high AURKA expression, and in patients with HNSCC.

e17520 Background: No biomarker of C resistance has been identified in HNSCC. PTEN loss is present in approximately 30% of HNSCC. Biomarker analysis of the E5397 study suggested that addition of C to cisplatin in R/M HNSCC improves PFS in PTEN high/PIK3CA wild type patients but not those with PTEN loss or PIK3CA mutation. We hypothesized that PTEN testing may aid patient (pt.) selection for C therapy in HNSCC. Methods: MCC15780 was a phase II randomized trial of C plus sorafenib or C plus placebo in R/M HNSCC. 52/56 pts. in this study received C. PTEN analysis using AQUA as previously described was performed on tumor from 38 pts. Automated image capture was performed with HistoRx PM-2000 using the AQUAsition software. AQUA PTEN cut off determined as 1177 based on the first tertile.Fisher’s exact test used to compare low and high expression group. Event-time distributions estimated by Kaplan-Meier and compared using log-rank. Stratified Cox proportional hazards models used to estimate hazard ratios (HR) and test for significance for OS and PFS. All p-values are two-sided. A level of p < 0.05 is considered statistically significant. Results: 12/37 (32%) tumors were PTEN low. There was statistically significant improvement in PFS in PTEN high tumors compared to PTEN low tumors (HR (high/low) =0.33, 95% CI= (0.14, 0.75), p=0.008); and this remains significant after adjusting for age, sex, race, and ethnicity (HR (high/low) =0.27, 95% CI= (0.11, 0.67), p=0.004). Conclusions: PFS is significantly longer in PTEN high tumors compared to PTEN low tumors (HR=0.33, 95% CI= (0.14-0.75), p=0.008) in patients with R/M HNSCC treated with C. This finding remains significant after adjusting for age, sex, race, and ethnicity (HR=0.27, 95% CI= (0.11-0.67), p=0.004).This warrants PTEN analysis of specimens from larger cetuximab-based RCT. [Table: see text]

Objectives. We compared faculty promotion rates by race/ethnicity across US academic medical centers. Methods. We used the Association of American Medical College's 1983 through 2000 faculty roster data to estimate median institution-specific promotion rates for assistant professor to associate professor and for associate professor to full professor. In unadjusted analyses, we compared medians for Hispanic and Black with White faculty using the Wilcoxon rank sum test. We compared institution-specific promotion rates between racial/ethnic groups with data stratified by institutional characteristic (institution size, proportion racial/ethnic minority faculty, and proportion women faculty) using the χ2 test. Our sample included 128 academic medical centers and 88 432 unique faculty. Results. The median institution-specific promotion rates for White, Hispanic, and Black faculty, respectively, were 30.2%, 23.5%, and 18.8% (P Conclusions. At most academic medical centers, promotion rates for Hispanic and Black were lower than those for White faculty. Equitable faculty promotion rates may reflect institutional climates that support the successful development of racial/ethnic minority trainees, ultimately improving healthcare access and quality for all patients.