David L. Crowe

Background/Aim: Prostate cancer is a common malignant tumor in men. DNA ligase IV (LIG4) expression correlated with poor prognosis in prostate cancer patients. LIG4 joins DNA double strand breaks and is essential for repair of these genetic lesions. Prostate cancers have not demonstrated clinically significant responses to anti-PD-1 immunotherapy. Prostate cancers express low PD-L1 levels and exhibit limited cytotoxic T lymphocyte infiltrates. To determine the effects of LIG4 inhibition on prostate tumorigenesis, we created a new genetically engineered in vivo model. Materials and Methods: Lig4+/+;TAg and Lig4+/−;TAg prostate glands and tumors were processed for histopathology. Separate groups of prostate tumor-bearing mice were treated with anti-PD1 antibody or preimmune IgG. LIG4 and PD-L1 expression was determined by quantitative reverse transcription polymerase chain reaction. Expression of DNA damage repair proteins, cell senescence, and cell death markers was determined by immunohistochemistry and immunofluorescence microscopy. The prostate cancer stem cell fraction was analyzed by Sca1/CD49f flow cytometry and tumorsphere culture. PD-L1 protein expression was determined by western blot. Results: LIG4 inhibition induced DNA double strand breaks and cellular senescence in prostate glands and cancers and significantly reduced prostate intraepithelial neoplasia and tumorigenesis. LIG4 inhibition reduced the prostate cancer stem cell fraction and proliferation in stem cell cultures. Prostate cancers resistant to LIG4 inhibition evaded anti-tumor immune response due to increased PD-L1 expression. PD-1 antibody treatment of these cancers induced CD8+ T lymphocyte infiltration and reduced tumor volume. Conclusion: Inhibition of LIG4 sensitized prostate cancers to immune checkpoint inhibition.

Background/Aim: The most frequently altered epigenetic modifier in head and neck squamous carcinoma (HNSC) is the histone methyltransferase KMT2D. KMT2D catalyzes methylation of histone H3K4 resulting in open chromatin and the activation of target genes. Tumor-associated macrophages (TAMs) promote cancer growth by causing T lymphocyte exhaustion. C-C motif chemokine ligand 2 (CCL2) is a potent TAM chemotactic factor. In HNSC, TAMs have been associated with unfavorable patient outcomes and metastasis. The aim of this study was to determine the role of KMT2D in HNSC using genetically engineered in vivo models. Materials and Methods: KMT2D protein expression was correlated with lymph node metastasis in human HNSC using immunohistochemistry. Genetically engineered KMT2D and CCL2 knockout models of HNSC were created in vivo. HNSC was characterized using qRT-PCR, histopathology, and immunohistochemistry/immunofluorescence microscopy. We also analyzed the effects of KMT2D expression on the proliferation and migration of human HNSC lines. The regulation of the CCL2 gene by KMT2D was characterized using chromatin immunoprecipitation-sequencing assay of transposase accessible chromatin-sequencing, and chromatin conformation capture-sequencing. Results: Human HNSC cases with high KMT2D expression exhibited significantly increased lymph node metastasis. Reduced KMT2D expression in our genetically engineered model correlated with reduced lymph node metastasis, longer latency, and slow tumor growth. CCL2 expression was decreased in KMT2D deficient HNSC, which correlated with a reduced TAM gene expression signature. Genomic experiments demonstrated that KMT2D directly targeted the CCL2 gene. A new genetically engineered in vivo model of CCL2-null HNSC was created, recapitulating the KMT2D deficient phenotype and showing a decreased T lymphocyte exhaustion signature. Conclusion: KMT2D regulates CCL2-mediated immune response and metastasis in HNSC.

The International Agency for Research on Cancer determined that obesity is the primary preventable cause of breast cancer. The nuclear receptor peroxisome proliferator activated receptor γ (PPARγ) binds inflammatory mediators in obesity and its expression is reduced in human breast cancer. We created a new model to better understand how the obese microenvironment alters nuclear receptor function in breast cancer. The obesity related cancer phenotype was PPARγ dependent; deletion of PPARγ in mammary epithelium which is a tumor suppressor in lean mice unexpectedly increased tumor latency, reduced the luminal progenitor (LP) tumor cell fraction, and increased autophagic and senescent cells. Loss of PPARγ expression in mammary epithelium of obese mice increased expression of 2-aminoadipate semialdehyde synthase (AASS) which regulates lysine catabolism to acetoacetate. PPARγ-associated co-repressors and activators regulated AASS expression via a canonical response element. AASS expression was significantly reduced in human breast cancer, and AASS overexpression or acetoacetate treatment inhibited proliferation and induced autophagy and senescence in human breast cancer cell lines. Genetic or pharmacologic HDAC inhibition promoted autophagy and senescence in mammary tumor cells in vitro and in vivo. We concluded that lysine metabolism is a novel metabolic tumor suppressor pathway in breast cancer.

Fig. S1. Telomere DNA damage is significantly increased in human prostate cancer. Fig. S2. Relative expression of TRF2 and p53 mRNAs in prostate glands from 8 week old K14Cre;TRF2+/+;p53+/+, K14Cre;TRF2f/f;p53+/+, and K14Cre;TRF2f/f;p53f/f mice is shown by qRT-PCR. Fig. S3. Increased senescence, decreased proliferation, and increased cell death in K14Cre;TRF2f/f;p53+/+ prostate epithelium. Fig. S4. Quantitation of SAHF+ cells in prostate glands from K14Cre;TRF2+/+;p53+/+, K14Cre;TRF2f/f;p53+/+, and K14Cre;TRF2f/f;p53f/f mice. Fig. S5. Defective secretion in K14Cre;TRF2f/f;p53+/+ prostate glands. Fig. S6. Telomere DNA damage signaling in K14Cre;TRF2f/f;p53+/+ prostate epithelium. Fig. S7. (A) Quantitation of 53BP1+ cells in prostate glands from K14Cre;TRF2+/+;p53+/+, K14Cre;TRF2f/f;p53+/+, and K14Cre;TRF2f/f;p53f/f mice. Fig. S8. Increased proliferation and reduced differentiation of K14Cre;TRF2f/f;TAg prostate tumors. Fig. S9. Decreased proliferation, increased AR+ differentiation, and apoptosis in K14Cre;TRF2f/f;Terc-/-;TAg prostate glands. Supplementary Table 1. Telomere DNA damage and tumor staging

Background: Head and neck cancer (HNC) is common worldwide. Given poor outcomes for patients with HNC, research into targeted therapies is needed. Ataxia telangiectasia mutated (ATM) is a DNA damage kinase which is activated by double-strand DNA breaks. We tested the effects of a novel ATM inhibitor on HNC cell lines and xenografts. Materials and Methods: p53-Binding protein 1 and phosphorylated ATM were localized in cultured cells by immunofluorescence microscopy. Protein expression was determined by western blot. Tumor xenografts were established by injecting HNC lines into immunocompromised mice. Tumor sections were characterized by immunohistochemistry. Apoptotic cells were determined by terminal transferase-mediated dUTP nick-end labeling assay. Results: ATM inhibition increased double-strand DNA breaks at replication foci in HNC cell lines. ATM inhibition affected cell-cycle regulatory protein expression, blocked cell-cycle progression at the G₂/M phase and resulted in apoptosis. Conclusion: ATM inhibition may be therapeutically useful in treating HNC.

Poly(ADP‐ribose) polymerases (PARP) act as DNA damage sensors that produce poly(ADP‐ribose) (PAR) chains at double‐strand breaks, facilitating the recruitment of repair factors. Cancers with homologous recombination defects are sensitive to small molecule PARP inhibitors. Despite PARP5B gene copy number changes in many cancers, the effects of this genetic alteration on tumor phenotype are largely unknown. To better understand this clinical finding, we characterized a PARP5B null mutation in a carcinogen‐induced in vivo head and neck squamous cell carcinoma (SCC) model. Reduced PARP5B expression inhibited tumor growth, induced primary tumor differentiation and apoptosis, and inhibited cell proliferation and metastasis. Loss of PARP5B expression‐induced ataxia telangiectasia and Rad3 related (ATR) activation and depleted the cancer stem cell fraction. PARP5B null tumor cells lacked 53BP1+ double‐strand break foci, ATM activation, and p53 induction compared to PARP5B+/+ cancers. PARP5B null SCC expresses a multiprotein complex containing PML, pRPA, Rad50, Rad51, XRCC1, proliferating cell nuclear antigen (PCNA), and Mcm2, suggesting an HR‐mediated repair mechanism at DNA replication foci. Low doses of etoposide combined with the PARP5B inhibitor XAV939 induced senescence and apoptosis in human SCC lines. NBS1 overexpression in these cells inhibited the effects of low‐dose etoposide/XAV939 treatment. Our results indicate that PARP5B inhibition is new targeted cancer therapy.

Telomere shortening has been demonstrated in benign prostatic hypertrophy (BPH), which is associated with prostate epithelial cell senescence. Telomere shortening is the most frequently observed genetic alteration in prostatic intraepithelial neoplasia, and is associated with poor clinical outcomes in prostate cancer. Gene expression database analysis revealed decreased TRF2 expression during malignant progression of the prostate gland. We reasoned that reduced TRF2 expression in prostate epithelium, by activating the telomere DNA damage response, would allow us to model both benign and malignant prostate disease. Prostate glands with reduced epithelial TRF2 expression developed age- and p53-dependent hypertrophy, senescence, ductal dilation, and smooth muscle hyperplasia similar to human BPH. Prostate tumors with reduced TRF2 expression were classified as high-grade androgen receptor–negative adenocarcinomas, which exhibited decreased latency, increased proliferation, and distant metastases. Prostate cancer stem cells with reduced TRF2 expression were highly tumorigenic and maintained telomeres both by telomerase and alternative lengthening (ALT). Telomerase inhibition in prostate glands with reduced TRF2 expression produced significant reduction in prostate tumor incidence by halting progression at intraepithelial neoplasia (PIN). These lesions were highly differentiated, exhibited low proliferation index, and high apoptotic cell fraction. Prostate tumors with reduced TRF2 expression and telomerase inhibition failed to metastasize and did not exhibit ALT.Implications: Our results demonstrate that the telomere DNA damage response regulates BPH, PIN, and prostate cancer and may be therapeutically manipulated to prevent prostate cancer progression.

Dyskeratosis congenita is a telomere DNA damage syndrome characterized by defective telomere maintenance, bone marrow failure, and increased head and neck cancer risk. The Pot1b−/−;Terc+/− mouse exhibits some features of dyskeratosis congenita, but head and neck cancer was not reported in this model. To model the head and neck cancer phenotype, we created unique Pot1b- and p53-null–mutant models which allow genetic lineage tracing of two distinct stem cell populations. Loss of Pot1b expression depleted stem cells via ATR/Chk1/p53 signaling. Tumorigenesis was inhibited in Pot1b−/−;p53+/+ mice due to cellular senescence. Pot1b−/−;p53−/− tumors also exhibited senescence, but proliferated and metastasized with expansion of Lgr6+ stem cells indicative of senescence-associated secretory phenotype. Selective depletion of the small K15+ stem cell fraction resulted in reduction of Lgr6+ cells and inhibition of tumorigenesis via senescence. Gene expression studies revealed that K15+ cancer stem cells regulate Lgr6+ cancer stem cell expansion via chemokine signaling. Genetic ablation of the chemokine receptor Cxcr2 inhibited cancer stem cell expansion and tumorigenesis via senescence. The effects of chemokines were primarily mediated by PI3K signaling, which is a therapeutic target in head and neck cancer.Implications: Paracrine interactions of cancer stem cell populations impact therapeutic options and patient outcomes.

Mammary gland luminal cells are maintained by the proliferation of ER− luminal progenitor (LP) cells. Human breast LP cells exhibit telomere DNA damage, which is associated with mammographic density and increased cancer risk. Telomeric repeat factor 2 (TRF2) protects telomeres from DNA damage response. TRF2 expression is reduced in human breast cancers. We deleted TRF2 expression in mammary gland epithelium. Mammary glands lacking TRF2 expression exhibited increased telomere DNA damage response, histopathological and functional degeneration, and prominent ductal fibrosis. TRF2‐deficient mammary tumors exhibited rapid onset and increased proliferation. Tumor derived LP cells failed to form tumors after transplantation. The MSC population was highly tumorigenic and maintained telomeres via the ALT mechanism. Telomere DNA damage response in mammary tumors resulted in p53 dependent ER+ cellular differentiation and sensitivity to anti‐estrogen therapy. Our results provide a new in vivo model of mammographic density, stem cell differentiation, cancer risk, and therapeutic sensitivity.