Germline Deficiency Research Articles

Ki67 deficiency impedes chromatin accessibility and BCR gene rearrangement.

The proliferation marker Ki67 has been attributed critical functions in maintaining mitotic chromosome morphology and heterochromatin organization during the cell cycle, indicating a potential role in developmental processes requiring rigid cell-cycle control. Here, we discovered that despite normal fecundity and organogenesis, germline deficiency in Ki67 resulted in substantial defects specifically in peripheral B and T lymphocytes. This was not due to impaired cell proliferation but rather to early lymphopoiesis at specific stages where antigen-receptor gene rearrangements occurred. We identified that Ki67 was required for normal global chromatin accessibility involving regulatory regions of genes critical for checkpoint stages in B cell lymphopoiesis. In line with this, mRNA expression of Rag1 was diminished and gene rearrangement was less efficient in the absence of Ki67. Transgenes encoding productively rearranged immunoglobulin heavy and light chains complemented Ki67 deficiency, completely rescuing early B cell development. Collectively, these results identify a unique contribution from Ki67 to somatic antigen-receptor gene rearrangement during lymphopoiesis.

Mismatch Repair (MMR) Gene Mutation Carriers Have Favorable Outcome in Colorectal and Endometrial Cancer: A Prospective Cohort Study.

Germline (Lynch syndrome, LS) and somatic deficiencies of mismatch repair proteins (MMRd) are linked to colorectal and endometrial cancer; however, their prognostic impact in Asian populations remains unclear. This prospective cohort study aimed to determine the prevalence and outcome of germline and somatic MMRd in cancer patients suspected of LS. Patients with colorectal or endometrial cancer suspected of LS were enrolled and underwent gene sequencing for germline MMRd (gMMRd) and immunohistochemistry staining of MMR proteins in a subset of the pathological samples (pMMRd). Among the 451 enrolled patients, 36 patients were gMMRd (+). Compared with gMMRd (-) patients, the 10-year relapse-free survival in gMMRd (+) patients was significantly higher (100% vs. 77.9%; p = 0.006), whereas the 10-year overall survival was similar (100% vs. 90.9%; p = 0.12). Among the 102 gMMRd (-) patients with available pMMR status, 13.7% were pMMRd (+). The 5-year relapse-free survival was 62.9% in gMMRd (-) pMMRd (+) patients and 35.0% in gMMRd (-) pMMRd (-) patients, both lower than gMMRd (+) patients (100%; p < 0.001). This study showed that having LS confers a favorable outcome in colorectal and endometrial cancer patients and highlights the importance of germline genetic testing following the detection of somatic MMRd.

Growth hormone-receptor disruption in mice reduces osteoarthritis and chondrocyte hypertrophy

Excessive growth hormone (GH) has been shown to promote joint degeneration in both preclinical and clinical studies. Little is known about the effect of disrupted GH or GH receptor (GHR) on joint health. The goal of this study is to investigate joint pathology in mice with either germline (GHR-/-) or adult inducible (iGHR-/-) GHR deficiency. Knee joints from male and female GHR-/- and WT mice at 24 months of age were processed for histological analysis. Also, knee joints from male and female iGHR-/- and WT mice at 22 months of age were scanned by micro-CT (μCT) for subchondral bone changes and characterized via histology for cartilage degeneration. Joint sections were also stained for the chondrocyte hypertrophy marker, COLX, and the cartilage degeneration marker, ADAMTS-5, using immunohistochemistry. Compared to WT mice, GHR-/- mice had remarkably smooth articular joint surfaces and an even distribution of proteoglycan with no signs of degeneration. Quantitatively, GHR-/- mice had lower OARSI and Mankin scores compared to WT controls. By contrast, iGHR-/- mice were only moderately protected from developing aging-associated OA. iGHR-/- mice had a significantly lower Mankin score compared to WT. However, Mankin scores were not significantly different between iGHR-/- and WT when males and females were analyzed separately. OARSI scores did not differ significantly between WT and iGHR-/- in either individual or combined sex analyses. Both GHR-/- and iGHR-/- mice had fewer COLX+ hypertrophic chondrocytes compared to WT, while no significant difference was observed in ADAMTS-5 staining. Compared to WT, a significantly lower trabecular thickness in the subchondral bone was observed in the iGHR-/- male mice but not in the female mice. However, there were no significant differences between WT and iGHR-/- mice in the bone volume to total tissue volume (BV/TV), bone mineral density (BMD), and trabecular number in either sex. This study identified that both germline and adult-induced GHR deficiency protected mice from developing aging-associated OA with more effective protection in GHR-/- mice.

Real world outcomes in patients with metastatic castration-resistant prostate cancer (mCRPC) treated with lutetium-177-PSMA-vipivotide tetraxetan (Lu177-PVT).

86 Background: Lu177-PVT is a novel radioligand, which was recently approved by the FDA to treat mCRPC. Real world data on its use and efficacy is limited. We aimed to build a new retrospective cohort of patients with mCRPC treated with 177Lu PSMA-617 in order to investigate clinical and pathological variables associated with treatment responses. Methods: We identified patients with a confirmed diagnosis of prostate cancer and were treated at Stanford University Medical Center with at least one dose of Lu177-PVT including trial patients. Data collection included demographic, clinicopathological and outcome variables. Statistical analysis was performed using Chi-squared and log-rank Kaplan-Meier analysis. Results: We identified 85 patients who met study criteria and were treated between March 2021 and June 2023, the median age was 66, 33% were non-white, median PSA at treatment initiation was 60, and 94% had osseous metastasis. Patients were pretreated with a median of six lines (range: 1-10) of prior therapy including local therapies; prostatectomy (28%), radiation (61%), brachytherapy (11%), taxane (78%), immunotherapy including checkpoint inhibitor and Sipuleucel-T (24%), PARP inhibitor (6%), and Radium-223 (15%). A majority of patients also received prior germline or somatic genetic testing (79%), with homologous recombination germline and somatic deficiencies present in 13% of patients. Of the identified cohort, 19 patients were still undergoing active treatment with Lu177-PVT at the time of data collection. Of the 66 patients not currently undergoing active Lu177-PVT treatment, 29 patients discontinued treatment due to progressive disease (44%) and 14 (21%) to toxicity related to 177Lu PSMA-617, completing a median of 4 cycles. 47 patients (71%) had an overall decline in their PSA from baseline, with 32 patients (49%) experiencing a decrease greater than fifty percent. Median overall survival of patients not actively receiving 177Lu PSMA-617 treatment was 366 days. Age, previous lines of therapy, and median time from diagnosis were not predictive of PSA treatment response (greater than fifty-percent reduction from baseline PSA). Conclusions: Lu177-PVT is a promising treatment for mCRPC in patients. Our results confirm ‘real-world’ activity of Lu177-PVT and align well with VISION study results, though real-world overall survival was comparatively shorter (12 months vs 15 months in VISION trial). Further investigation is warranted into predictors of response and toxicity.

Phenotypic Characterization of Male Tafazzin-Knockout Mice at 3, 6, and 12 Months of Age

Barth syndrome (BTHS) is an X-linked mitochondrial disease caused by mutations in the gene encoding for tafazzin (TAZ), a key enzyme in the remodeling of cardiolipin. Mice with a germline deficiency in Taz have been generated (Taz-KO) but not yet fully characterized. We performed physiological assessments of 3-, 6-, and 12-month-old male Taz-KO mice, including measures of perinatal survival, growth, lifespan, gross anatomy, whole-body energy and substrate metabolism, glucose homeostasis, and exercise capacity. Taz-KO mice displayed reduced viability, with lower-than-expected numbers of mice recorded at 4 weeks of age, and a shortened lifespan due to disease progression. At all ages, Taz-KO mice had lower body weights compared with wild-type (Wt) littermates despite similar absolute food intakes. This finding was attributed to reduced adiposity and diminutive organs and tissues, including heart and skeletal muscles. Although there were no differences in basal levels of locomotion between age-matched genotypes, indirect calorimetry studies showed higher energy expenditure measures and respiratory exchange ratios in Taz-KO mice. At the youngest age, Taz-KO mice had comparable glucose tolerance and insulin action to Wt mice, but while these measures indicated metabolic impairments in Wt mice with advancing age that were likely associated with increasing adiposity, Taz-KO mice were protected. Comparisons across the three age-cohorts revealed a significant and more severe deterioration of exercise capacity in Taz-KO mice than in their Wt littermate controls. The Taz-KO mouse model faithfully recapitulates important aspects of BTHS, and thus provides an important new tool to investigate pathophysiological mechanisms and potential therapies.

Development of an interactive precision medicine dashboard to aid in completion of germline and homologous recombination deficiency (HRD) tumor testing for newly diagnosed ovarian cancer patients to improve the use of PARP inhibitor frontline maintenance (583)

Development of an interactive precision medicine dashboard to aid in completion of germline and homologous recombination deficiency (HRD) tumor testing for newly diagnosed ovarian cancer patients to improve the use of PARP inhibitor frontline maintenance (583)

708 Aire deficiency induces upregulation of JAK-STAT signaling in keratinocytes and results in alopecia areata-like lesions in mice

Alopecia areata (AA) is an autoimmune hair loss disorder with no cure. We report here that C57BL/6J female mice with germline deficiency in autoimmune regulator (Aire-/-) spontaneously developed AA-like lesions (n=61/98; 62%). Aire-/- lesions exhibited AA hallmarks such as miniaturized hair follicles (HFs), decreased hair width (-29%, p<0.0173) and length (-27%, p=0.0002), and increased lymphocytic infiltrate at the hair bulb (+191% in CD8+ T cells, p=0.0546) relative to body site matched Aire+/+ tissue.

An antagonistic pleiotropic gene regulates the reproduction and longevity tradeoff

The antagonistic pleiotropy theory of aging proposes that genes enhancing fitness in early life limit the lifespan, but the molecular evidence remains underexplored. By profiling translatome changes in Caenorhabditis elegans during starvation recovery, we find that an open reading frame (ORF) trl-1 “hidden” within an annotated pseudogene significantly translates upon refeeding. trl-1 mutant animals increase brood sizes but shorten the lifespan and specifically impair germline deficiency–induced longevity. The loss of trl-1 abnormally up-regulates the translation of vitellogenin that produces copious yolk to provision eggs, whereas vitellogenin overexpression is known to reduce the lifespan. We show that the TRL-1 protein undergoes liquid–liquid phase separation (LLPS), through which TRL-1 granules recruit vitellogenin messenger RNA and inhibit its translation. These results indicate that trl-1 functions as an antagonistic pleiotropic gene to regulate the reproduction–longevity tradeoff by optimizing nutrient production for the next generation.

Genetic Perturbation of CD70/CD27 Co-Stimulation Promotes the Development of Bcl6-Driven Diffuse Large B-Cell Lymphoma

Multiple co-stimulatory and co-inhibitory pathways modulate T-cell dependent anti-tumor immune responses in lymphoid malignancies. We recently defined the recurrent genetic alterations and associated substructure of diffuse large B-cell lymphoma (DLBCL), including five distinct clusters (1-5), and identified potential genetic bases for immune evasion [Nature Medicine 2018; 24:679-690]. In our series, 26% of tumors had inactivating somatic mutations or copy loss of CD70 and likely disruption of CD70/CD27 co-stimulation.CD70 and CD27 are homotrimer type II and homodimer type I transmembrane proteins and members of the TNF and TNF receptor superfamilies, respectively. CD70 is transiently expressed on certain normal B-cell and dendritic cell populations upon activation and constitutively expressed on multiple B-cell tumors. CD70 activation of CD27 + T cells promotes antigen-dependent T-cell expansion and immune surveillance of normal and malignant B cells. Patients with germline deficiencies of either CD70 or CD27 have an increased incidence of EBV-associated lymphoid malignancies, underscoring the importance of this co-stimulatory pathway.In our series, CD70 alterations were most common in Cluster 1 DLBCLs, which also exhibited recurrent BCL6 chromosomal translocations. The co-occurrence of CD70 and BCL6 genetic alterations was noteworthy because of the established role of CD8 + T-cell dependent immune surveillance in murine models of Bcl6-driven DLBCL [Nature Medicine 2014; 20:283-290]. To assess the consequences of Cd70 deficiency and perturbed CD70/CD27 co-stimulation on Bcl6-driven lymphomagenesis, we crossed the previously described Bcl6tg/+ and Cd70-/- mice to generate Cd70-/-; Bcl6tg/+ animals. In the aging cohorts, Cd70-/-; Bcl6tg/+ mice were more likely than Bcl6tg/+ animals (or the Cd70-/- or wild-type [WT] groups) to be euthanized for symptoms before the study endpoint (18 months [mo]) (5 of 18 Cd70-/-; Bcl6tg/+ euthanized for symptoms prior to the first of 9 Bcl6tg/+). Additionally, significantly greater total numbers and percentages of Cd70-/-; Bcl6tg/+ mice, in comparison to Bcl6tg/+ or Cd70-/- animals, were euthanized for symptoms (64% Cd70-/-; Bcl6tg/+ vs. 29% Bcl6tg/+, p=0.005 and 64% Cd70-/-; Bcl6tg/+ vs. 11% Cd70-/-, p=0.002). Almost all euthanized Cd70-/; Bcl6tg/+ and Bcl6tg/+ micehad massively enlarged spleens infiltrated with histopathologically confirmed DLBCLs characterized by clonal Ig gene rearrangements. Our findings indicate that genetic perturbation of Cd70 accelerates the onset and significantly increases the incidence of Bcl6-driven DLBCL.To characterize potential differences in the anti-tumor immune responses in Cd70-/-; Bcl6tg/+ and Bcl6tg/+ mice (and Cd70-/- and WT controls), we also harvested spleens from 6 animals in each of the aging cohorts at predetermined intervals (2, 6, 14 and 18 mo). We analyzed the composition of splenic-cell suspensions by flow cytometry and evaluated the intact splenic architecture and morphology with expert hematopathologists (VS, KW and SR). At 14 and 18 mo, spleens from WT and Cd70-/- animals were largely normal in appearance and size. In contrast, spleens from 14 and 18 mo Bcl6tg/+ and Cd70-/-; Bcl6tg/+ mice exhibited disordered architecture and abnormal pre-malignant lymphoid proliferation with expanded white pulp including morphologically and immunophenotypically aberrant B cells of small to large size and increased infiltrating T-cells. Additionally, significantly higher fractions of splenic CD8 + T cells from 14 and 18 mo Bcl6tg/+ and Cd70-/-; Bcl6tg/+ animals expressed the CD69 activation marker and exhibited terminal differentiation, consistent with an ongoing anti-tumor immune response. Interestingly, Bcl6tg/+ animals had significantly higher percentages of splenic TCRβ + T cells at the earlier time point (14 mo) with delayed-onset splenomegaly (at 18 mo), which is in marked contrast to the Cd70-/-; Bcl6tg/+ mice that had significantly lower percentages of splenic TCRβ + T cells at the earlier time point (14 mo) and early-onset splenomegaly (14 mo). These findings suggest that the initial T-cell mediated immune response was more effective in Bcl6tg/+ than Cd70-/-; Bcl6tg/+ animals. Taken together, our data indicate that genetic perturbation of CD70/CD27 co-stimulation limits the development of an effective anti-tumor immune response in Bcl6tg/+-driven DLBCL. DisclosuresNeuberg: Madrigal Pharmaceuticals: Other: Stock ownership; Pharmacyclics: Research Funding. Rodig: Affimed: Research Funding; Bristol-Myers-Squibb: Research Funding; Merck: Research Funding; Immunitas: Membership on an entity's Board of Directors or advisory committees; KITE/Gilead: Research Funding. Shipp: Bristol Myers Squibb: Research Funding; Immunitas Therapeutics: Consultancy; AstraZeneca: Consultancy, Research Funding; Merck: Research Funding; Abbvie: Other: Institution: Research Grant/Funding; Bayer: Other: Institution: Research Grant/Funding.

Germline POT1 Deregulation Can Predispose to Myeloid Malignancies in Childhood.

While the shelterin complex guards and coordinates the mechanism of telomere regulation, deregulation of this process is tightly linked to malignant transformation and cancer. Here, we present the novel finding of a germline stop-gain variant (p.Q199*) in the shelterin complex gene POT1, which was identified in a child with acute myeloid leukemia. We show that the cells overexpressing the mutated POT1 display increased DNA damage and chromosomal instabilities compared to the wildtype counterpart. Protein and mRNA expression analyses in the primary patient cells further confirm that, physiologically, the variant leads to a nonfunctional POT1 allele in the patient. Subsequent telomere length measurements in the primary cells carrying heterozygous POT1 p.Q199* as well as POT1 knockdown AML cells revealed telomeric elongation as the main functional effect. These results show a connection between POT1 p.Q199* and telomeric dysregulation and highlight POT1 germline deficiency as a predisposition to myeloid malignancies in childhood.

Saturated very long chain fatty acid configures glycosphingolipid for lysosome homeostasis in long-lived C. elegans

The contents of numerous membrane lipids change upon ageing. However, it is unknown whether and how any of these changes are causally linked to lifespan regulation. Acyl chains contribute to the functional specificity of membrane lipids. In this study, working with C. elegans, we identified an acyl chain-specific sphingolipid, C22 glucosylceramide, as a longevity metabolite. Germline deficiency, a conserved lifespan-extending paradigm, induces somatic expression of the fatty acid elongase ELO-3, and behenic acid (22:0) generated by ELO-3 is incorporated into glucosylceramide for lifespan regulation. Mechanistically, C22 glucosylceramide is required for the membrane localization of clathrin, a protein that regulates membrane budding. The reduction in C22 glucosylceramide impairs the clathrin-dependent autophagic lysosome reformation, which subsequently leads to TOR activation and longevity suppression. These findings reveal a mechanistic link between membrane lipids and ageing and suggest a model of lifespan regulation by fatty acid-mediated membrane configuration.

NF-κB c-Rel Is Dispensable for the Development but Is Required for the Cytotoxic Function of NK Cells.

Natural Killer (NK) cells are cytotoxic lymphocytes critical to the innate immune system. We found that germline deficiency of NF-κB c-Rel results in a marked decrease in cytotoxic function of NK cells, both in vitro and in vivo, with no significant differences in the stages of NK cell development. We found that c-Rel binds to the promoters of perforin and granzyme B, two key proteins required for NK cytotoxicity, and controls their expression. We generated a NK cell specific c-Rel conditional knockout to study NK cell intrinsic role of c- Rel and found that both global and conditional c-Rel deficiency leads to decreased perforin and granzyme B expression and thereby cytotoxic function. We also confirmed the role of c-Rel in perforin and granzyme B expression in human NK cells. c-Rel reconstitution rescued perforin and granzyme B expressions in c-Rel deficient NK cells and restored their cytotoxic function. Our results show a previously unknown role of c-Rel in transcriptional regulation of perforin and granzyme B expressions and control of NK cell cytotoxic function.

DNA Polymerase and Mismatch Repair Exert Distinct Microsatellite Instability Signatures in Normal and Malignant Human Cells.

Although replication repair deficiency, either by mismatch repair deficiency (MMRD) and/or loss of DNA polymerase proofreading, can cause hypermutation in cancer, microsatellite instability (MSI) is considered a hallmark of MMRD alone. By genome-wide analysis of tumors with germline and somatic deficiencies in replication repair, we reveal a novel association between loss of polymerase proofreading and MSI, especially when both components are lost. Analysis of indels in microsatellites (MS-indels) identified five distinct signatures (MS-sigs). MMRD MS-sigs are dominated by multibase losses, whereas mutant-polymerase MS-sigs contain primarily single-base gains. MS deletions in MMRD tumors depend on the original size of the MS and converge to a preferred length, providing mechanistic insight. Finally, we demonstrate that MS-sigs can be a powerful clinical tool for managing individuals with germline MMRD and replication repair-deficient cancers, as they can detect the replication repair deficiency in normal cells and predict their response to immunotherapy. SIGNIFICANCE: Exome- and genome-wide MSI analysis reveals novel signatures that are uniquely attributed to mismatch repair and DNA polymerase. This provides new mechanistic insight into MS maintenance and can be applied clinically for diagnosis of replication repair deficiency and immunotherapy response prediction.This article is highlighted in the In This Issue feature, p. 995.

Clinical implications of mismatch repair deficiency in prostate cancer.

Immune checkpoint blockade holds great promise in the treatment of solid tumors but has not yet been approved for use in advanced prostate cancer. This is largely due to the relatively modest response in clinical trials in unselected patients and the lack of available biomarkers to predict clinical benefit. Germline and somatic mismatch repair (MMR)gene deficiencies are more prevalent than previously thought, especially in the metastatic setting, in patients with high-grade Gleason scores and in patients with variant histologies. An early signal suggests that patients with deficiency in MMR may respond well to immunotherapy. Both germline and somatic genetic testing are recommended, yet questions remain on the best modality for testing given lack of standardization and false-negative results in patients with complex genomic structural rearrangements. Expanded panels, such as next generation sequencing may increase the sensitivity without compromising specificity. Future studies are still needed to explore the relationships of hypermutation, tumor mutational burden, tumor-infiltrating lymphocytes and microsatellite instability-H status as predictors of response to immunotherapy. The drivers of variable response is largely unknown, and a more mature understanding of the mechanisms of resistance in deficiencies in MMR tumors may help to more precisely inform use of immunotherapy in prostate cancer.

The Heterogeneity Between Lynch-Associated and Sporadic MMR Deficiency in Colorectal Cancers.

Previous studies demonstrated that prognosis of germline deficiency in mismatch repair protein (dMMR) was different from that of sporadic dMMR. The underlying mechanism has not been studied. From a prospectively maintained database, we collected dMMR colorectal cancer (CRC) patients identified by postoperative immunohistochemistry screening. According to genetic test, patients were grouped as Lynch-associated or sporadic dMMR. We compared the clinical-pathological features, prognosis, and immunoreactive differences between the two groups. By whole-exome sequencing and neoantigen detection pipeline, mutational frequencies and neoantigen burdens were also compared. All statistical tests were two-sided. Sixty-seven sporadic dMMR and 85 Lynch-associated CRC patients were included in the study. Sporadic dMMR patients were older (P < .001) and their tumors were poorly differentiated (P = .03). The survival was better in the Lynch-associated group (P = .001). After adjustment, the difference still remained statistically significant (hazard ratio = 0.29, 95% confidence interval = 0.09 to 0.95, P = .04). The scores of Crohn's-like reaction (CRO; P < .001), immunoreactions in the invasive margin (IM; P = .01), tumor stroma (TS; P = .009), and cancer nest (CN; P = .02) of the Lynch-associated group were statistically significantly higher. The numbers of CD3+, CD8+, Foxp3+ tumor-infiltrating lymphocytes (TILs) in IM; CD3+, CD4+ TILs in TS; and CD3+, CD4+, CD8+ TILs in CN were statistically significantly higher in Lynch-associated dMMR patients. Based on the 16 patients who under went whole-exome sequencing, there were also more somatic mutations and neoantigen burdens in the Lynch-associated group compared with the sporadic dMMR group (439/pt vs 68/pt, P = .006; 628/pt vs 97/pt, P = .009). There are heterogeneities in dMMR CRCs. Lynch-associated dMMR patients present with more somatic mutations and neoantigens compared with sporadic dMMR, which probably results in stronger immunoreactions and survival improvement.

The STK35 locus contributes to normal gametogenesis and encodes a lncRNA responsive to oxidative stress.

ABSTRACTSerine/threonine kinase 35 (STK35) is a recently identified human kinase with an autophosphorylation function, linked functionally to actin stress fibers, cell cycle progression and survival. STK35 has previously been shown to be highly expressed in human testis, and we demonstrated its regulation by nuclear-localized importin α2 in HeLa cells. The present study identifies progressive expression from the STK35 locus of two coding mRNA isoforms and one long non-coding RNA (lncRNA) in mouse testis during spermatogenesis, indicating their tightly controlled synthesis. Additionally, lncRNA transcripts are increased by exposure to oxidative stress in mouse GC-1 germ cell line. STK35 knockout (KO) mice lacking all three RNAs are born at sub-Mendelian frequency, and adults manifest both male and female germline deficiency. KO males exhibit no or partial spermatogenesis in most testis tubule cross-sections; KO ovaries are smaller and contain fewer follicles. Eyes of KO mice display phenotypes ranging from gross deformity to mild goniodysgenesis or iridocorneal angle malformation, to overtly normal. These findings demonstrate the tight regulation of transcription from the STK35 locus and its central importance to fertility, eye development and cell responses to oxidative stress.

Abstract 4987: ASS1 downregulation facilitates cancer cell survival in acidic environment

Abstract ”pH gradient reversal”, namely the alkalinization of intracellular pH (pHi) and acidification of extracellular pH (pHe), is a unique phenotype of cancer cells which enable cancer survival, migration and invasion. Hence, not surprisingly, pH gradient reversal is emerging as a new hallmark of cancer. Argininosuccinate synthase, ASS1, is a urea cycle enzyme found to be downregulated by methylation in multiple different types of cancers in correlation with increased metastasis and poor prognosis. We recently showed that ASS1 downregulation increases proliferation by channeling aspartate towards pyrimidine synthesis by CAD. We now show that under hypoxia and acidic environments, ASS1 is downregulated together with CAD by HIF1α as part of an essential network necessary for cancer cells’ survival, invasion and migration. Similar to germline deficiency of ASS1 which increases the levels of glutamine and ammonia, ASS1 downregulation by HIF1αenables the cancer cells to increase glutamine levels allowing its utilization for glutathione and importantly, for preservation of alkali pHi by elevating ammonia levels and buffering the acidity caused by cancers’ enhanced glycolysis and the consequent increase in lactic acid. Moreover, cancer cells which express ASS1 when grown in culture, downregulate its expression in hypoxic regions when grown in vivo, suggesting that these cancers might also be sensitive to drugs previously utilized only in ASS1 deficient cancers. Finally, we show that treating cancers with ASS1 deficiency with ammonia scavengers as given to ASS1 deficient patients, increases apoptosis and restricts migration, and is hence beneficial therapeutically. In summary, ASS1 downregulation is regulated differently at altered carcinogenic states, promoting proliferation as well as cancer survival and metastasis. Citation Format: Ayelet Erez, Alon Silberman, Adi Jacob, Shiran Limanovich, Odeya Boukobza1, Erez Persi, Rom Keshet, Alona Sarver, Joo Sang Lee, Alexander Brandis, Keren Bahar Halpern, Raya Eilam, Ben Pode, Yair Anikster, Igor Ulitsky, Sandesh Nagamani, Eytan Ruppin. ASS1 downregulation facilitates cancer cell survival in acidic environment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4987. doi:10.1158/1538-7445.AM2017-4987

Extracellular Signal-Regulated Kinase Signaling in CD4-Expressing Cells Inhibits Osteochondromas.

Defects in cartilage homeostasis can give rise to various skeletal disorders including osteochondromas. Osteochondromas are benign bone tumors caused by excess accumulation of chondrocytes, the main cell type of cartilage. The extracellular signal-regulated kinase (ERK) pathway is a major signaling node that functions within chondrocytes to regulate their growth and differentiation. However, it is not known whether the ERK pathway in other cell types regulates cartilage homeostasis. We show here that mice with a germline deficiency of Erk1 and a conditional deletion of Erk2 in cells that express CD4, or expressed CD4 at one point in development, unexpectedly developed bone deformities. The bone lesions were due to neoplastic outgrowths of chondrocytes and disordered growth plates, similar to tumors observed in the human disease, osteochondromatosis. Chondrocyte accumulation was not due to deletion of Erk2 in the T cells. Rather, CD4cre was expressed in cell types other than T cells, including a small fraction of chondrocytes. Surprisingly, the removal of T cells accelerated osteochondroma formation and enhanced disease severity. These data show for the first time that T cells impact the growth of osteochondromas and describe a novel model to study cartilage homeostasis and osteochondroma formation.

The emerging role of homologous recombination repair and PARP inhibitors in genitourinary malignancies.

As cells age and are exposed to genotoxic stress, preservation of the genomic code requires multiple DNA repair pathways to remove single-strand or double-strand breaks. Loss of function somatic genomic aberrations or germline deficiency in genes involved in DNA repair can result in acute cell death or, after a latency period, cellular transformation. Therapeutic exploitation of DNA repair by inhibition of poly (adenosine diphosphate [ADP]) ribose polymerases (PARP), a family of enzymes involved in the repair of single-strand and in some cases double-strand breaks, has become a novel cancer treatment. Although the application of PARP inhibitors (PARPis) initially focused on tumors with BRCA1 or BRCA2 deficiencies, synthetic susceptibilities to PARPis have been expanded due to the identification of tumors with mutations pathways involved in DNA damage repair, in particular those that repair double-strand breaks using homologous recombination (HR). There is an increasing appreciation that genitourinary (GU) malignancies, including bladder cancer and especially prostate cancer, contain subsets of patients with germline and somatic alterations in HR genes that may reflect an increased response to PARPis. In this review, the authors describe the mechanisms and rationale of the use of PARPis in patients with GU cancers, summarize previously reported preclinical and clinical trials, and identify ongoing trials to determine how PARPis and strategies targeted at HR repair can have widespread application in patients with GU cancers. Cancer 2017;123:1912-1924. © 2017 American Cancer Society.

Key Role of STAT4 Deficiency in the Hematopoietic Compartment in Insulin Resistance and Adipose Tissue Inflammation.

Visceral adipose tissue (AT) inflammation is linked to the complications of obesity, including insulin resistance (IR) and type 2 diabetes. Recent data from our lab showed that germline deficiency in STAT4 reduces inflammation and improves IR in obese mice. The objective of this study was to determine the contribution of selective STAT4 deficiency in subsets of hematopoietic cells to IR and AT inflammation. To determine the contribution of hematopoietic lineage, we sublethally irradiated Stat4−/−C57Bl6 mice and reconstituted them with bone marrow cells (BMC) from Stat4+/+C57Bl6 congenic donors. We also established the contribution of selective STAT4 deficiency in CD4+ or CD8+ T cells using adoptive transfer in Rag1−/− mice. All mice received a HFD for 15 weeks (n = 7–12 mice/group). BMC that expressed STAT4 induced increases in glucose intolerance and IR compared to STAT4-deficient cells. Also, AT inflammation was increased and the numbers of CD8+ cells infiltrating AT were higher in mice with STAT4 expressing BMC. Studies in Rag1−/− mice further confirmed the prominent role of CD8+ cells expressing STAT4 in insulin resistance and AT and islet inflammation. Collectively our results show specific and dominant contribution of STAT4 in the hematopoietic compartment to metabolic health and inflammation in diet-induced obesity.