Histone demethylase KDM5D upregulation drives sex differences in colon cancer.

Sex exerts a profound impact on cancer incidence, spectrum and outcomes, yet the molecular and genetic bases of such sex differences are ill-defined and presumptively ascribed to X-chromosome genes and sex hormones1. Such sex differences are particularly prominent in colorectal cancer (CRC) in which men experience higher metastases and mortality. A murine CRC model, engineered with an inducible transgene encoding oncogenic mutant KRASG12D and conditional null alleles of Apc and Trp53 tumour suppressors (designated iKAP)2, revealed higher metastases and worse outcomes specifically in males with oncogenic mutant KRAS (KRAS*) CRC. Integrated cross-species molecular and transcriptomic analyses identified Y-chromosome gene histone demethylase KDM5D as a transcriptionally upregulated gene driven by KRAS*-mediated activation of the STAT4 transcription factor. KDM5D-dependent chromatin mark and transcriptome changes showed repression of regulators of the epithelial cell tight junction and major histocompatibility complex class I complex components. Deletion of Kdm5d in iKAP cancer cells increased tight junction integrity, decreased cell invasiveness and enhanced cancer cell killing by CD8+ T cells. Conversely, iAP mice engineered with a Kdm5d transgene to provide constitutive Kdm5d expression specifically in iAP cancer cells showed an increased propensity for more invasive tumours in vivo. Thus, KRAS*-STAT4-mediated upregulation of Y chromosome KDM5D contributes substantially to the sex differences in KRAS* CRC by means of its disruption of cancer cell adhesion properties and tumour immunity, providing an actionable therapeutic strategy for metastasis risk reduction for men afflicted with KRAS* CRC.

PKM2 isoform-specific deletion reveals a differential requirement for pyruvate kinase in tumor cells.

The pyruvate kinase M2 isoform (PKM2) is expressed in cancer and plays a role in regulating anabolic metabolism. To determine whether PKM2 is required for tumor formation or growth, we generated mice with a conditional allele that abolishes PKM2 expression without disrupting PKM1 expression. PKM2 deletion accelerated mammary tumor formation in a Brca1-loss-driven model of breast cancer. PKM2 null tumors displayed heterogeneous PKM1 expression, with PKM1 found in nonproliferating tumor cells and no detectable pyruvate kinase expression in proliferating cells. This suggests that PKM2 is not necessary for tumor cell proliferation and implies that the inactive state of PKM2 is associated with the proliferating cell population within tumors, whereas nonproliferating tumor cells require active pyruvate kinase. Consistent with these findings, variable PKM2 expression and heterozygous PKM2 mutations are found in human tumors. These data suggest that regulation of PKM2 activity supports the different metabolic requirements of proliferating and nonproliferating tumor cells.

Telomerase reactivation following telomere dysfunction yields murine prostate tumors with bone metastases.

To determine the role of telomere dysfunction and telomerase reactivation in generating pro-oncogenic genomic events and in carcinoma progression, an inducible telomerase reverse transcriptase (mTert) allele was crossed onto a prostate cancer-prone mouse model null for Pten and p53 tumor suppressors. Constitutive telomerase deficiency and associated telomere dysfunction constrained cancer progression. In contrast, telomerase reactivation in the setting of telomere dysfunction alleviated intratumoral DNA-damage signaling and generated aggressive cancers with rearranged genomes and new tumor biological properties (bone metastases). Comparative oncogenomic analysis revealed numerous recurrent amplifications and deletions of relevance to human prostate cancer. Murine tumors show enrichment of the TGF-ß/SMAD4 network, and genetic validation studies confirmed the cooperative roles of Pten, p53, and Smad4 deficiencies in prostate cancer progression, including skeletal metastases. Thus, telomerase reactivation in tumor cells experiencing telomere dysfunction enables full malignant progression and provides a mechanism for acquisition of cancer-relevant genomic events endowing new tumor biological capabilities.

Insulin Receptor-Expressing T Cells Appear in Individuals at Risk for Type 1 Diabetes and Can Move into the Pancreas in C57BL/6 Transgenic Mice.

Insulin receptor (IR) expression on the T cell surface can indicate an activated state; however, the IR is also chemotactic, enabling T cells with high IR expression to physically move toward insulin. In humans with type 1 diabetes (T1D) and the NOD mouse model, a T cell-mediated autoimmune destruction of insulin-producing pancreatic ß cells occurs. In previous work, when purified IR+ and IR- T cells were sorted from diabetic NOD mice and transferred into irradiated nondiabetic NOD mice, only those that received IR+ T cells developed insulitis and diabetes. In this study, peripheral blood samples from individuals with T1D (new onset to 14 y of duration), relatives at high-risk for T1D, defined by positivity for islet autoantibodies, and healthy controls were examined for frequency of IR+ T cells. High-risk individuals had significantly higher numbers of IR+ T cells as compared with those with T1D (p < 0.01) and controls (p < 0.001); however, the percentage of IR+ T cells in circulation did not differ significantly between T1D and control subjects. With the hypothesis that IR+ T cells traffic to the pancreas in T1D, we developed a (to our knowledge) novel mouse model exhibiting a FLAG-tagged mouse IR on T cells on the C57BL/6 background, which is not susceptible to developing T1D. Interestingly, these C57BL/6-CD3FLAGmIR/mfm mice showed evidence of increased IR+ T cell trafficking into the islets compared with C57BL/6 controls (p < 0.001). This transgenic animal model provides a (to our knowledge) novel platform for investigating the influence of IR expression on T cell trafficking and the development of insulitis.

Effective combinatorial immunotherapy for castration-resistant prostate cancer.

A significant fraction of patients with advanced prostate cancer treated with androgen deprivation therapy experience relapse with relentless progression to lethal metastatic castration-resistant prostate cancer (mCRPC). Immune checkpoint blockade using antibodies against cytotoxic-T-lymphocyte-associated protein 4 (CTLA4) or programmed cell death 1/programmed cell death 1 ligand 1 (PD1/PD-L1) generates durable therapeutic responses in a significant subset of patients across a variety of cancer types. However, mCRPC showed overwhelming de novo resistance to immune checkpoint blockade, motivating a search for targeted therapies that overcome this resistance. Myeloid-derived suppressor cells (MDSCs) are known to play important roles in tumour immune evasion. The abundance of circulating MDSCs correlates with prostate-specific antigen levels and metastasis in patients with prostate cancer. Mouse models of prostate cancer show that MDSCs (CD11b+Gr1+) promote tumour initiation and progression. These observations prompted us to hypothesize that robust immunotherapy responses in mCRPC may be elicited by the combined actions of immune checkpoint blockade agents together with targeted agents that neutralize MDSCs yet preserve T-cell function. Here we develop a novel chimaeric mouse model of mCRPC to efficiently test combination therapies in an autochthonous setting. Combination of anti-CTLA4 and anti-PD1 engendered only modest efficacy. Targeted therapy against mCRPC-infiltrating MDSCs, using multikinase inhibitors such as cabozantinib and BEZ235, also showed minimal anti-tumour activities. Strikingly, primary and metastatic CRPC showed robust synergistic responses when immune checkpoint blockade was combined with MDSC-targeted therapy. Mechanistically, combination therapy efficacy stemmed from the upregulation of interleukin-1 receptor antagonist and suppression of MDSC-promoting cytokines secreted by prostate cancer cells. These observations illuminate a clinical path hypothesis for combining immune checkpoint blockade with MDSC-targeted therapies in the treatment of mCRPC.

Telomerase reactivation reverses tissue degeneration in aged telomerase-deficient mice.

An ageing world population has fuelled interest in regenerative remedies that may stem declining organ function and maintain fitness. Unanswered is whether elimination of intrinsic instigators driving age-associated degeneration can reverse, as opposed to simply arrest, various afflictions of the aged. Such instigators include progressively damaged genomes. Telomerase-deficient mice have served as a model system to study the adverse cellular and organismal consequences of wide-spread endogenous DNA damage signalling activation in vivo. Telomere loss and uncapping provokes progressive tissue atrophy, stem cell depletion, organ system failure and impaired tissue injury responses. Here, we sought to determine whether entrenched multi-system degeneration in adult mice with severe telomere dysfunction can be halted or possibly reversed by reactivation of endogenous telomerase activity. To this end, we engineered a knock-in allele encoding a 4-hydroxytamoxifen (4-OHT)-inducible telomerase reverse transcriptase-oestrogen receptor (TERT-ER) under transcriptional control of the endogenous TERT promoter. Homozygous TERT-ER mice have short dysfunctional telomeres and sustain increased DNA damage signalling and classical degenerative phenotypes upon successive generational matings and advancing age. Telomerase reactivation in such late generation TERT-ER mice extends telomeres, reduces DNA damage signalling and associated cellular checkpoint responses, allows resumption of proliferation in quiescent cultures, and eliminates degenerative phenotypes across multiple organs including testes, spleens and intestines. Notably, somatic telomerase reactivation reversed neurodegeneration with restoration of proliferating Sox2(+) neural progenitors, Dcx(+) newborn neurons, and Olig2(+) oligodendrocyte populations. Consistent with the integral role of subventricular zone neural progenitors in generation and maintenance of olfactory bulb interneurons, this wave of telomerase-dependent neurogenesis resulted in alleviation of hyposmia and recovery of innate olfactory avoidance responses. Accumulating evidence implicating telomere damage as a driver of age-associated organ decline and disease risk and the marked reversal of systemic degenerative phenotypes in adult mice observed here support the development of regenerative strategies designed to restore telomere integrity.

The differentiation and stress response factor XBP-1 drives multiple myeloma pathogenesis.

Multiple myeloma (MM) evolves from a highly prevalent premalignant condition termed MGUS. The factors underlying the malignant transformation of MGUS are unknown. We report a MGUS/MM phenotype in transgenic mice with Emu-directed expression of the XBP-1 spliced isoform (XBP-1s), a factor governing unfolded protein/ER stress response and plasma-cell development. Emu-XBP-1s elicited elevated serum Ig and skin alterations. With age, Emu-xbp-1s transgenics develop features diagnostic of human MM, including bone lytic lesions and subendothelial Ig deposition. Furthermore, transcriptional profiles of Emu-xbp-1s lymphoid and MM cells show aberrant expression of known human MM dysregulated genes. The similarities of this model with the human disease, coupled with documented frequent XBP-1s overexpression in human MM, serve to implicate XBP-1s dysregulation in MM pathogenesis.

Overlapping functions of Hdac1 and Hdac2 in cell cycle regulation and haematopoiesis.

Histone deacetylases (HDACs) counterbalance acetylation of lysine residues, a protein modification involved in numerous biological processes. Here, Hdac1 and Hdac2 conditional knock-out alleles were used to study the function of class I Hdac1 and Hdac2 in cell cycle progression and haematopoietic differentiation. Combined deletion of Hdac1 and Hdac2, or inactivation of their deacetylase activity in primary or oncogenic-transformed fibroblasts, results in a senescence-like G(1) cell cycle arrest, accompanied by up-regulation of the cyclin-dependent kinase inhibitor p21(Cip). Notably, concomitant genetic inactivation of p53 or p21(Cip) indicates that Hdac1 and Hdac2 regulate p53-p21(Cip)-independent pathways critical for maintaining cell cycle progression. In vivo, we show that Hdac1 and Hdac2 are not essential for liver homeostasis. In contrast, total levels of Hdac1 and Hdac2 in the haematopoietic system are critical for erythrocyte-megakaryocyte differentiation. Dual inactivation of Hdac1 and Hdac2 results in apoptosis of megakaryocytes and thrombocytopenia. Together, these data indicate that Hdac1 and Hdac2 have overlapping functions in cell cycle regulation and haematopoiesis. In addition, this work provides insights into mechanism-based toxicities observed in patients treated with HDAC inhibitors.

A BAC transgenic reporter recapitulates in vivo regulation of human telomerase reverse transcriptase in development and tumorigenesis.

Telomerase is tightly regulated in humans relative to mice, owing to the differential regulation of TERT genes. To explore hTERT regulation in vivo, we engineered mice with a 160-kb transgenic bacterial artificial chromosome (BAC) spanning the hTERT locus with a Renilla luciferase (Rluc) cassette downstream of its promoter. Analysis of multiple founder lines revealed that the Rluc expression profile from the transgenic hTERT reporter locus reproduced that of the native hTERT gene in all tissues and organs examined, demonstrating that genetic sequence determined the species-specific developmental regulation of the hTERT gene and that mouse epigenetic and transcription machineries faithfully regulated hTERT transcription. Thus, these mice allowed detailed analyses of developmental hTERT regulation. Both the transgenic hTERT reporter and the endogenous mTERT locus were expressed in early embryonic stages, and their mRNA levels progressively decreased throughout embryonic and postnatal development. Whereas hTERT transcription was much lower than mTERT expression in most organs, it increased significantly during postnatal development of thymus, testis, and ovary. In testis, the Rluc mRNA was enriched in elongating spermatids of seminiferous tubules. In addition, the transcription of transgenic hTERT reporter, but surprisingly not the endogenous mTERT gene, was activated during Wnt1-induced mammary tumorigenesis, allowing the monitoring of tumor development via noninvasive bioluminescent imaging. Collectively, our results demonstrate that the hTERT transgenic reporter system recapitulates the developmental regulation of the hTERT gene in a chromosomal position-independent manner and serves as a legitimate model to explore telomerase regulation in the development of normal and neoplastic tissues in vivo.

Pyruvate Kinase M1 Suppresses Development and Progression of Prostate Adenocarcinoma.

SIGNIFICANCE: Differential expression of PKM1 and PKM2 impacts prostate tumorigenesis and suggests a potential therapeutic vulnerability in prostate cancer.

Telomerase Reverse Transcriptase Preserves Neuron Survival and Cognition in Alzheimer's Disease Models.

Amyloid-induced neurodegeneration plays a central role in Alzheimer's disease (AD) pathogenesis. Here, we show that telomerase reverse transcriptase (TERT) haploinsufficiency decreases BDNF and increases amyloid-ß (Aß) precursor in murine brain. Moreover, prior to disease onset, the TERT locus sustains accumulation of repressive epigenetic marks in murine and human AD neurons, implicating TERT repression in amyloid-induced neurodegeneration. To test the impact of sustained TERT expression on AD pathobiology, AD mouse models were engineered to maintain physiological levels of TERT in adult neurons, resulting in reduced Aß accumulation, improved spine morphology, and preserved cognitive function. Mechanistically, integrated profiling revealed that TERT interacts with ß-catenin and RNA polymerase II at gene promoters and upregulates gene networks governing synaptic signaling and learning processes. These TERT-directed transcriptional activities do not require its catalytic activity nor telomerase RNA. These findings provide genetic proof-of-concept for somatic TERT gene activation therapy in attenuating AD progression including cognitive decline.

Chromatin Regulator CHD1 Remodels the Immunosuppressive Tumor Microenvironment in PTEN-Deficient Prostate Cancer.

Genetic inactivation of PTEN is common in prostate cancer and correlates with poorer prognosis. We previously identified CHD1 as an essential gene in PTEN-deficient cancer cells. Here, we sought definitive in vivo genetic evidence for, and mechanistic understanding of, the essential role of CHD1 in PTEN-deficient prostate cancer. In Pten and Pten/Smad4 genetically engineered mouse models, prostate-specific deletion of Chd1 resulted in markedly delayed tumor progression and prolonged survival. Chd1 deletion was associated with profound tumor microenvironment (TME) remodeling characterized by reduced myeloid-derived suppressor cells (MDSC) and increased CD8+ T cells. Further analysis identified IL6 as a key transcriptional target of CHD1, which plays a major role in recruitment of immunosuppressive MDSCs. Given the prominent role of MDSCs in suppressing responsiveness to immune checkpoint inhibitors (ICI), our genetic and tumor biological findings support combined testing of anti-IL6 and ICI therapies, specifically in PTEN-deficient prostate cancer. SIGNIFICANCE: We demonstrate a critical role of CHD1 in MDSC recruitment and discover CHD1/IL6 as a major regulator of the immunosuppressive TME of PTEN-deficient prostate cancer. Pharmacologic inhibition of IL6 in combination with immune checkpoint blockade elicits robust antitumor responses in prostate cancer.This article is highlighted in the In This Issue feature, p. 1241.

Class IA phosphoinositide 3-kinase regulates heart size and physiological cardiac hypertrophy.

Class I(A) phosphoinositide 3-kinases (PI3Ks) are activated by growth factor receptors, and they regulate, among other processes, cell growth and organ size. Studies using transgenic mice overexpressing constitutively active and dominant negative forms of the p110alpha catalytic subunit of class I(A) PI3K have implicated the role of this enzyme in regulating heart size and physiological cardiac hypertrophy. To further understand the role of class I(A) PI3K in controlling heart growth and to circumvent potential complications from the overexpression of dominant negative and constitutively active proteins, we generated mice with muscle-specific deletion of the p85alpha regulatory subunit and germ line deletion of the p85beta regulatory subunit of class I(A) PI3K. Here we show that mice with cardiac deletion of both p85 subunits exhibit attenuated Akt signaling in the heart, reduced heart size, and altered cardiac gene expression. Furthermore, exercise-induced cardiac hypertrophy is also attenuated in the p85 knockout hearts. Despite such defects in postnatal developmental growth and physiological hypertrophy, the p85 knockout hearts exhibit normal contractility and myocardial histology. Our results therefore provide strong genetic evidence that class I(A) PI3Ks are critical regulators for the developmental growth and physiological hypertrophy of the heart.

Cooperative interactions of p53 mutation, telomere dysfunction, and chronic liver damage in hepatocellular carcinoma progression.

Hepatocellular carcinoma is among the most common and lethal cancers in humans. Hepatocellular carcinoma is commonly associated with physical or functional inactivation of the p53 tumor suppressor, high levels of chromosomal instability, and disease conditions causing chronic cycles of hepatocyte death and regeneration. Mounting evidence has implicated regeneration-induced telomere erosion as a potential mechanism fueling genome instability. In mouse models of hepatocellular carcinoma, telomere dysfunction has been shown to enhance initiation of hepatic neoplasias yet constrain full malignant progression of these neoplasms possibly due to activation of a p53-dependent checkpoint and/or intolerable levels of genomic instability. Here, in a hepatocellular carcinoma-prone model brought about through toxin-induced hepatocyte injury and regeneration, we sought to determine the cooperative interactions of germ line p53 mutation and telomere dysfunction [produced by telomerase reverse transcriptase (mTERT) gene knockout]. In the setting of intact telomeres, p53 mutation had no effect on hepatocarcinogenesis, whereas in the setting of telomere dysfunction, p53 mutation enabled advanced hepatocellular carcinoma disease. Notably, there was no evidence of deletion or mutation of the wild-type p53 allele in the late generation mTert(-/-)p53(+/-) mice, suggesting that reduced levels of p53 potently enable hepatocellular carcinoma progression in the setting of telomere dysfunction. Thus, this study supports a model that, in the face of chronic liver damage, attenuated p53 function and telomere-induced chromosomal instability play critical and cooperative roles in the progression of hepatocellular carcinoma.

Exogenous Cushing syndrome with inhaled fluticasone in a child receiving lopinavir/ritonavir.

OBJECTIVE: To describe a case of Cushing syndrome in a child during concurrent use of inhaled fluticasone propionate, nasal mometasone, and a highly active antiretroviral regimen including lopinavir/ritonavir. CASE SUMMARY: A 9-year-old boy with HIV infection and asthma developed moon facies, increased facial hair, and increased weight after fluticasone propionate inhalation (1 puff; 220 microg) therapy was begun. His antiretroviral regimen contained the protease inhibitor combination lopinavir/ritonavir at a dose of 216/54 mg twice daily, and he had been stable for the previous 5 years. He had also been receiving intranasal mometasone for 11 months for the management of allergic rhinitis. Serum cortisol and adrenocorticotropic hormone levels were consistent with adrenal suppression. These physical findings and symptoms and laboratory values normalized after discontinuation of the fluticasone propionate. The Naranjo probability scale indicated that a probable interaction occurred between lopinavir/ritonavir and fluticasone propionate, leading to subsequent adrenal suppression. DISCUSSION: Protease inhibitors are associated with numerous drug interactions due to inhibition of the CYP3A4 isoenzyme. Pharmaceutical agents used to treat comorbidities in HIV-infected individuals often can interact with protease inhibitors, leading to toxic drug concentrations or untoward effects. Inhaled corticosteroids such as fluticasone propionate are often necessary to treat asthma in young children and are metabolized by CYP3A4. Interactions between protease inhibitors and inhaled fluticasone propionate have been reported in the adult population, but reports are limited in the pediatric literature. CONCLUSIONS: This case raises awareness of the interaction between fluticasone propionate and lopinavir/ritonavir and adverse effects in children receiving both medications.

Qki deficiency maintains stemness of glioma stem cells in suboptimal environment by downregulating endolysosomal degradation.

Stem cells, including cancer stem cells (CSCs), require niches to maintain stemness, yet it is unclear how CSCs maintain stemness in the suboptimal environment outside their niches during invasion. Postnatal co-deletion of Pten and Trp53 in mouse neural stem cells (NSCs) leads to the expansion of these cells in their subventricular zone (SVZ) niches but fails to maintain stemness outside the SVZ. We discovered that Qki is a major regulator of NSC stemness. Qk deletion on a Pten-/-; Trp53-/- background helps NSCs maintain their stemness outside the SVZ in Nes-CreERT2; QkL/L; PtenL/L; Trp53L/L mice, which develop glioblastoma with a penetrance of 92% and a median survival time of 105 d. Mechanistically, Qk deletion decreases endolysosome-mediated degradation and enriches receptors essential for maintaining self-renewal on the cytoplasmic membrane to cope with low ligand levels outside niches. Thus, downregulation of endolysosome levels by Qki loss helps glioma stem cells (GSCs) maintain their stemness in suboptimal environments outside their niches.

Development of Resistance to EGFR-Targeted Therapy in Malignant Glioma Can Occur through EGFR-Dependent and -Independent Mechanisms.

Epidermal growth factor receptor (EGFR) is highly amplified, mutated, and overexpressed in human malignant gliomas. Despite its prevalence and growth-promoting functions, therapeutic strategies to inhibit EGFR kinase activity have not been translated into profound beneficial effects in glioma clinical trials. To determine the roles of oncogenic EGFR signaling in gliomagenesis and tumor maintenance, we generated a novel glioma mouse model driven by inducible expression of a mutant EGFR (EGFR*). Using combined genetic and pharmacologic interventions, we revealed that EGFR*-driven gliomas were insensitive to EGFR tyrosine kinase inhibitors, although they could efficiently inhibit EGFR* autophosphorylation in vitro and in vivo. This is in contrast with the genetic suppression of EGFR* induction that led to significant tumor regression and prolonged animal survival. However, despite their initial response to genetic EGFR* extinction, all tumors would relapse and propagate independent of EGFR*. We further showed that EGFR*-independent tumor cells existed prior to treatment and were responsible for relapse following genetic EGFR* suppression. And, the addition of a PI3K/mTOR inhibitor could significantly delay relapse and prolong animal survival. Our findings shed mechanistic insight into EGFR drug resistance in glioma and provide a platform to test therapies targeting aberrant EGFR signaling in this setting.

Dual Roles of RNF2 in Melanoma Progression.

UNLABELLED: Epigenetic regulators have emerged as critical factors governing the biology of cancer. Here, in the context of melanoma, we show that RNF2 is prognostic, exhibiting progression-correlated expression in human melanocytic neoplasms. Through a series of complementary gain-of-function and loss-of-function studies in mouse and human systems, we establish that RNF2 is oncogenic and prometastatic. Mechanistically, RNF2-mediated invasive behavior is dependent on its ability to monoubiquitinate H2AK119 at the promoter of LTBP2, resulting in silencing of this negative regulator of TGFß signaling. In contrast, RNF2's oncogenic activity does not require its catalytic activity nor does it derive from its canonical gene repression function. Instead, RNF2 drives proliferation through direct transcriptional upregulation of the cell-cycle regulator CCND2. We further show that MEK1-mediated phosphorylation of RNF2 promotes recruitment of activating histone modifiers UTX and p300 to a subset of poised promoters, which activates gene expression. In summary, RNF2 regulates distinct biologic processes in the genesis and progression of melanoma via different molecular mechanisms. SIGNIFICANCE: The role of epigenetic regulators in cancer progression is being increasingly appreciated. We show novel roles for RNF2 in melanoma tumorigenesis and metastasis, albeit via different mechanisms. Our findings support the notion that epigenetic regulators, such as RNF2, directly and functionally control powerful gene networks that are vital in multiple cancer processes.