Despite its critical role in survival, many aspects of hematopoiesis remain unresolved. In the classical model of the hematopoietic program, quiescent hematopoietic stem cells (HSCs) sit at the top of the hematopoietic hierarchy, with the ability to self-renew and differentiate as needed. HSCs give rise to more proliferative progenitor cells, which possess multipotent potential, but have largely or completely lost self-renewal capabilities. Here, we have identified the tumor suppressor, Inhibitor of Growth 4 (Ing4), as a critical regulator of multipotent progenitor (MPP) homeostasis. In the absence of Ing4, we show that MPPs express a transcriptional program of hematopoietic activation, yet they remain quiescent with low levels of reactive oxygen species. Functionally, Ing4-deficient MPPs are capable of robust regeneration following competitive bone marrow transplantation, resulting in substantially higher blood chimerism compared to wild-type MPPs. These data suggest Ing4 deficiency promotes a poised state in MPPs, quiescent, but transcriptionally primed for activation, and capable of converting the poised state into robust repopulation upon stress. Our model provides key tools for further identification and characterization of pathways that control quiescence and regeneration in MPPs.

The pygmy sperm whale (Kogia breviceps) possesses an exocrine gland associated with its false gill slit pigmentation pattern. The cervical gill slit gland is a compound tubuloalveolar gland that produces a holocrine secretion and displays maturational changes in size and secretory histology. While the morphology of the cervical gill slit gland has been described in detail, to date, the chemical composition of its secretion remains uncharacterized. This study used histochemical staining techniques and quantitative lipid analysis to identify and characterize the constituents expressed in the secretory cells and secretion of the cervical gill slit gland. Results demonstrate that the secretion, like those of terrestrial artiodactyls that function in chemical communication, includes a complex mixture of carbohydrates, proteins, and lipids. Differences in staining intensity across germinal and secretory epithelial layers demonstrate differential expression, or maturation, of mucins and proteins. Additionally, a highly unusual and primary constituent of the secretion is uric acid. Uric acid was identified within the secretion using histochemical stains and polarized light imaging, and chemically verified using scanning electron microscopy with energy dispersive spectrometry. While uric acid is not a common constituent of mammalian exocrine glands, urate-based compounds are abundant in the secretions of marine organisms used in chemical communication. Thus, uric acid may contribute to the chemical message produced by K. breviceps in its marine environment. We hypothesize that the chemical signals produced by the gill slit gland may be shared at close-range by conspecifics, and that the mode of sensory reception is likely gustation.

Lymph node involvement and copy number alterations by race

Brain metastases frequently develop in patients with non-small cell lung cancer (NSCLC) and are a common cause of cancer-related deaths, yet our understanding of the underlying human biology is limited. Here we performed multimodal single-nucleus RNA and T cell receptor, single-cell spatial and whole-genome sequencing of brain metastases and primary tumors of patients with treatment-naive NSCLC. Chromosomal instability (CIN) is a distinguishing genomic feature of brain metastases compared with primary tumors, which we validated through integrated analysis of molecular profiling and clinical data in 4,869 independent patients, and a new cohort of 12,275 patients with NSCLC. Unbiased analyses revealed transcriptional neural-like programs that strongly enriched in cancer cells from brain metastases, including a recurring, CINhigh cell subpopulation that preexists in primary tumors but strongly enriched in brain metastases, which was also recovered in matched single-cell spatial transcriptomics. Using multiplexed immunofluorescence in an independent cohort of treatment-naive pairs of primary tumors and brain metastases from the same patients with NSCLC, we validated genomic and tumor-microenvironmental findings and identified a cancer cell population characterized by neural features strongly enriched in brain metastases. This comprehensive analysis provides insights into human NSCLC brain metastasis biology and serves as an important resource for additional discovery.

Differential DNA methylation by race

Advanced HNC differential mutations and gene expression by race

Squamous cell cancers (SCCs) of the head and neck, esophagus, and lung, referred to as aero-upper digestive SCCs, are prevalent in the United States and worldwide. Their incidence and mortality are projected to increase at alarming rates, posing diagnostic, prognostic, and therapeutic challenges. These SCCs share certain epigenetic, genomic, and genetic alterations, immunologic properties, environmental exposures, as well as lifestyle and nutritional risk factors, which may underscore common complex gene-environmental interactions across them. This review focuses upon the frequent shared epigenetic, genomic, and genetic alterations, emerging preclinical model systems, and how this collective knowledge can be leveraged into perspectives on standard of care therapies and mechanisms of resistance, nominating new potential directions in translational therapeutics.

Oropharyngeal HNC subtype mutations and copy number alterations by race

HNC patient cohort IDs and datasets

Differentially expressed genes by race

Abstract INTRODUCTION: Metastasis is associated with more than 90% of cancer-related mortality, and thus, there is a compelling need for innovative therapeutic breakthroughs. TP53 mutations are present in 60-70% of human cancers, especially in squamous cell cancers. For example, TP53 mutations are detected in up to 80% of esophageal squamous cell carcinomas (ESCCs). Accumulating evidence suggests that certain missense mutant forms of p53 such as R175 (R172 in mouse), R273 and R282 can acquire neomorphic pro-oncogenic activities that are referred to as gain-of-function (GOF). To elucidate novel mutant p53-dependent mechanisms in promoting metastasis, we conducted RNA-Seq, p53 ChIP-Seq and H3K27ac CUT&RUN-Seq on primary and metastatic tumor cells harvested from our mouse model of ESCC harboring Trp53R172H/−, Trp53−/− and Trp53+/+. Herein, we have identified p53-R172H gene targets in metastatic ESCC. One of the dominant effectors of p53-R172H-mediated metastasis was Colony stimulating factor-1 (Csf-1) signaling through its cognate receptor (Csf-1r), which we recently published in Cancer Discovery (PMID: 37676642). Overall, this study aims to investigate the GOF properties and mediators of mutant p53 in promoting ESCC metastasis. RESULTS/DISCUSSION: Using ex vivo cultures and in vivo tail-vein injections, we demonstrated increased colonization and metastatic capabilities of ESCC cells harboring p53-R172H in comparison to the tumor cells with null and wild-type p53. p53-R172H occupies more genes in the metastatic ESCCs than in the primary tumors that is independent of p53 expression levels. We identified 72 unique targets of p53-R172H with altered gene occupancy and expression that occurs during the transition from primary tumor to metastasis. Such targets in metastatic ESCC are exemplified by Csf-1 and Birc5 (encoding anti-apoptosis protein Survivin) that are also enriched with H3K27ac in their promoters. They are upregulated during metastasis compared to the primary tumors dependent upon p53 mutation status, which is reinforced by TCGA data and ESCC patient-derived tissue microarrays (TMAs). Interestingly, other frequently detected “hotspot” p53 mutations including R282W and R273C, which we introduced into the ESCC cells using base editing promote differential pro-tumorigenic activities. Indeed, the analysis of SCC datasets in TCGA reveals that specific p53 mutations are associated with altered overall survival, as well as differences in the enriched pathways. CONCLUSION: We have demonstrated GOF properties and mediators of p53-R172H in promoting ESCC metastasis that may be applicable to other SCCs. We are further expanding our analyses to additional “hotspot” p53 mutations to dissect the mechanisms of distinct p53 mutations in fostering ESCC metastasis, which can open up new avenues for therapeutic applications. Citation Format: Gizem Efe, Katherine Cunningham, Raul Navaridas Fernandez de Bobadilla, Karen J. Dunbar, Kensuke Sugiura, Noriyuki Nishiwaki, Saul Carcamo, Lois Resnick-Silverman, Dan Hasson, Andres J. Klein-Szanto, Alison M. Taylor, James J. Manfredi, Carol Prives, Anil K. Rustgi. p53-R172H mutation confers gain-of-function properties and promotes metastasis in squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1270.

Abstract A major challenge in osteosarcoma (OS) immunotherapy is overcoming the immunosuppressive tumor microenvironment (TME), to restore immune recognition and destruction of tumor cells. One of the key immune cell subsets known to contribute to the immunosuppressive TME are M2-polarized tumor-associated macrophages (TAMs). Our work aims to identify novel oncolytic virus (OV) combination immunotherapies to induce direct lysis of OS tumor cells, activate immune-mediated destruction of OS, and remodel the immunosuppressive TME. The direct lytic activity of oncolytic herpes simplex virus (oHSV; HSV1716) treatment was assessed using LIVE/DEAD flow cytometry and viral replication was measured using standard plaque assay. NK cell degranulation and killing assays were performed by co-culture of OS cell lines with peripheral blood mononuclear cells (PBMC) treated ± oHSV; NK cell degranulation and target cell death was assessed by flow cytometry. TAMs were generated in vitro by co-culture of PBMC with OS cell lines or primary bone marrow-derived mesenchymal stem cells (MSCs) for 7 days. Phagocytosis assays were performed by co-culture of TAMs with OS cells using fluorescent cell tracker stains and flow cytometry. Multicellular spheroids were generated by co-culture of firefly luciferase-expressing OS cell lines with CD14+ monocytes and bone marrow derived MSCs for 7 days in low adhesion plates, viability of tumor cells after treatment was assessed by addition of d-luciferin and quantification of luminescence. Treatment of four OS cell lines with oHSV for 72 hours resulted in <20% cell death by direct lysis. However, after treatment with oHSV-GFP, >95% of OS cells were GFP+ and viral titer increased up to 105-fold when compared to viral input. This highlighted high levels of infection and replication of oHSV within OS cell lines, despite limited oncolysis. Moreover, oHSV treatment of PBMC significantly enhanced the immune-mediated killing of OS cell lines, and this was NK cell dependent. Monolayer co-culture of PBMC with OS cell lines or MSC generated TAMs with significantly increased pro-tumor M2-like phenotype (CD14+CD206+CD163+), when compared with PBMC cultured alone, and TAMs suppressed NK cell degranulation against OS cell lines. Treatment of TAMS with oHSV encoding GM-CSF reduced the proportion of M2 TAMs. Moreover, treatment of OS target cells with anti-EGFR monoclonal antibody (mAb) and co-culture with in vitro generated TAMs significantly increased their phagocytic activity. Multicellular spheroids incorporating MSCs and CD14+ monocytes displayed a high degree of resistance against immune-mediated killing, when compared with OS cell line alone as monolayer or spheroid cultures. However, combination of OV with anti-EGFR mAb significantly increased immune-mediated killing against multicellular spheroids. oHSV combination strategies with mAb may be a promising treatment strategy against OS, to exert direct oncolysis, stimulate immune-mediated destruction, and remodel the immunosuppressive TME. Citation Format: Tyler K. Barr, Victoria A. Jennings, Alison Taylor, Jessica Murby, Natasha J. Caplen, Javed Khan, Richard Baugh, Heather E. Owston, Dennis McGonagle, Fiona Errington-Mais, Graham P. Cook. Oncolytic HSV1716-GMCSF combination strategies to remodel the immunosuppressive osteosarcoma tumor-microenvironment and promote anti-tumor immunity [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pediatric Cancer Research; 2024 Sep 5-8; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl):Abstract nr B049.

Abstract Deleterious mutations in STK11/LKB1, KEAP1, and SMARCA4 have been shown to be associated with adverse response to immunotherapy in several lung cancer cohorts. However, the biological and clinical implications of deletions of these genes, which all reside on chromosome 19p (chr19p), has not been extensively described. We sought to first characterize the association between immune infiltration and these copy number changes in chr19p. To do this, we analyzed mutation, copy number, and gene expression data from non-small cell lung cancer cohorts in the Cancer Genome Atlas (TCGA). Chr19p is frequently deleted across cancers in the TCGA dataset, with higher rates in lung adenocarcinoma. In both lung adenocarcinoma and squamous cell carcinoma, nearly half of TCGA tumors have deletion of chr19p including STK11, KEAP1, and SMARCA4, and these tumors are enriched for STK11 mutation. Immune infiltrate (estimated by methylation patterns) was not significantly associated with STK11 mutation but was significantly reduced in tumors with chr19 deletion when controlling for tumor type and overall copy number load. In addition, low-level deletion of any of these genes correlated with decreased expression of immune signaling pathways. Based on these findings, we next wanted to characterize the association between chr19p copy number and immunotherapy response. We previously reported that low-level deletion of STK11 correlated with an immunosuppressive, treatment-refractory phenotype in samples collected from non-small cell lung cancer (NSCLC) patients with resectable disease in a phase II study of neoadjuvant atezolizumab + chemotherapy. To confirm these findings in a larger cohort, we examined the AACR GENIE NSCLC biopharma collective (BPC) dataset. A subset of this cohort included patients treated with immunotherapy (atezolizumab, nivolumab, and/or pembrolizumab) and with copy number data available or GISTIC analysis of individual genes (n = 204). Here, deletion of any of these three genes correlated with worse overall survival, and this was statistically significant for STK11 deletion. Overall, our data suggests that a broader set of patients, defined by copy number changes in these genes, may experience this adverse immunobiology. Further study to elucidate the mechanistic implications of this association is warranted. Citation Format: Brian S. Henick, Yohanna Georgis, Benjamin O. Herzberg, Carla P. Concepcion-Crisol, Alison M. Taylor. Copy number alterations in chromosome 19 genes associate with reduced immune infiltrate and adverse outcomes in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3634.

Abstract This abstract is being presented as a short talk in the scientific program. A full abstract is printed in the Proffered Abstracts section (PR016) of the Conference Program/Proceedings. Citation Format: Nadja Zhakula-Kostadinova, Sejal Jain, Laura Byron, Matthew L. Meyerson, Alison M. Taylor. Investigating vulnerabilities associated with chromosome arm aneuploidy in cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr B018.

PURPOSE In lung squamous cell carcinoma (LUSC), Black patients show significantly higher incidence and lower overall survival than White patients. Although socioeconomic factors likely contribute to this survival disparity, genomic factors have yet to be elucidated in LUSC. METHODS Using 416 LUSC tumor samples in the Cancer Genome Atlas (TCGA), we assessed genomic and transcriptomic profiles by ancestry. We replicated our analyses in pan-cancer data from TCGA, the American Association of Cancer Research (AACR) Genomics Evidence Neoplasia Information Exchange (GENIE), and Columbia University Medical Center. RESULTS We found increased MYC amplification, LUSC-specific MYC enhancer amplification, and chromosome arm 8q (chr8q) gain to be significantly associated with genetic AFR (African) ancestry in LUSC in TCGA. Furthermore, expression of MYC target genes was significantly enriched in AFR samples. Local ancestry analysis identified correlation of chr8q gain with AFR ancestry at the MYC locus in TCGA. We also found a significant correlation between chr8q and AFR ancestry in multiple cancer types and pan-cancer in TCGA. Similarly, in a pan-cancer subset of AACR GENIE data, we found a significant correlation between chr8q gain and race. CONCLUSION Together, our data suggest that ancestry may influence amplification of not only MYC but also its enhancer in LUSC. They also suggest a role for genetic ancestry in chr8q aneuploidy in cancer. These studies further define and expand patients who may benefit from future anti- MYC therapeutic approaches.