Senescence-Induced Vascular Remodeling Creates Therapeutic Vulnerabilities in Pancreas Cancer.

KRAS mutant pancreatic ductal adenocarcinoma (PDAC) is characterized by a desmoplastic response that promotes hypovascularity, immunosuppression, and resistance to chemo- and immunotherapies. We show that a combination of MEK and CDK4/6 inhibitors that target KRAS-directed oncogenic signaling can suppress PDAC proliferation through induction of retinoblastoma (RB) protein-mediated senescence. In preclinical mouse models of PDAC, this senescence-inducing therapy produces a senescence-associated secretory phenotype (SASP) that includes pro-angiogenic factors that promote tumor vascularization, which in turn enhances drug delivery and efficacy of cytotoxic gemcitabine chemotherapy. In addition, SASP-mediated endothelial cell activation stimulates the accumulation of CD8+ T cells into otherwise immunologically "cold" tumors, sensitizing tumors to PD-1 checkpoint blockade. Therefore, in PDAC models, therapy-induced senescence can establish emergent susceptibilities to otherwise ineffective chemo- and immunotherapies through SASP-dependent effects on the tumor vasculature and immune system.

Sexually dimorphic tumor suppression by small mitochondrial Arf.

Internal translational initiation of the mRNA encoding the Arf tumor suppressor yields an N-terminally truncated small Arf protein (smArf) that lacks amino acid residues required for Mdm2 binding and p53 activation. Here, we report that female, but not male, mice engineered to produce only smArf in lieu of the full-length Arf protein retain residual, sexually dimorphic tumor suppressive activity.

NK cell-mediated cytotoxicity contributes to tumor control by a cytostatic drug combination.

Molecularly targeted therapies aim to obstruct cell autonomous programs required for tumor growth. We show that mitogen-activated protein kinase (MAPK) and cyclin-dependent kinase 4/6 inhibitors act in combination to suppress the proliferation of KRAS-mutant lung cancer cells while simultaneously provoking a natural killer (NK) cell surveillance program leading to tumor cell death. The drug combination, but neither agent alone, promotes retinoblastoma (RB) protein-mediated cellular senescence and activation of the immunomodulatory senescence-associated secretory phenotype (SASP). SASP components tumor necrosis factor-α and intercellular adhesion molecule-1 are required for NK cell surveillance of drug-treated tumor cells, which contributes to tumor regressions and prolonged survival in a KRAS-mutant lung cancer mouse model. Therefore, molecularly targeted agents capable of inducing senescence can produce tumor control through non-cell autonomous mechanisms involving NK cell surveillance.

Mouse medulloblastoma driven by CRISPR activation of cellular Myc.

MYC-driven Group 3 (G3) medulloblastoma (MB) is the most aggressive of four molecular subgroups classified by transcriptome, genomic landscape and clinical outcomes. Mouse models that recapitulate human G3 MB all rely on retroviral vector-induced Myc expression driven by viral regulatory elements (Retro-Myc tumors). We used nuclease-deficient CRISPR/dCas9-based gene activation with combinatorial single guide RNAs (sgRNAs) to enforce transcription of endogenous Myc in Trp53-null neurospheres that were orthotopically transplanted into the brains of naïve animals. Three combined sgRNAs linked to dCas9-VP160 induced cellular Myc expression and large cell anaplastic MBs (CRISPR-Myc tumors) which recapitulated the molecular characteristics of mouse and human G3 MBs. The BET inhibitor JQ1 suppressed MYC expression in a human G3 MB cell line (HD-MB03) and CRISPR-Myc, but not in Retro-Myc MBs. This G3 MB mouse model in which Myc expression is regulated by its own promoter will facilitate pre-clinical studies with drugs that regulate Myc transcription.

Small mitochondrial Arf (smArf) protein corrects p53-independent developmental defects of Arf tumor suppressor-deficient mice.

The mouse p19Arf (human p14ARF) tumor suppressor protein, encoded in part from an alternative reading frame of the Ink4a (Cdkn2a) gene, inhibits the Mdm2 E3 ubiquitin ligase to activate p53. Arf is not expressed in most normal tissues of young mice but is induced by high thresholds of aberrant hyperproliferative signals, thereby activating p53 in incipient tumor cells that have experienced oncogene activation. The single Arf mRNA encodes two distinct polypeptides, including full-length p19Arf and N-terminally truncated and unstable p15smArf ("small mitochondrial Arf") initiated from an internal in-frame AUG codon specifying methionine-45. Interactions of p19Arf with Mdm2, or separately with nucleophosmin (NPM, B23) that localizes and stabilizes p19Arf within the nucleolus, require p19Arf N-terminal amino acids that are not present within p15smArf We have generated mice that produce either smARF alone or M45A-mutated (smArf-deficient) full-length p19Arf proteins. BCR-ABL-expressing pro/pre-B cells producing smArf alone are as oncogenic as their Arf-null counterparts in generating acute lymphoblastic leukemia when infused into unconditioned syngeneic mice. In contrast, smArf-deficient cells from mice of the ArfM45A strain are as resistant as wild-type Arf+/+ cells to comparable oncogenic challenge and do not produce tumors. Apart from being prone to tumor development, Arf-null mice are blind, and their male germ cells exhibit defects in meiotic maturation and sperm production. Although ArfM45A mice manifest the latter defects, smArf alone remarkably rescues both of these p53-independent developmental phenotypes.

Inactivation of Ezh2 Upregulates Gfi1 and Drives Aggressive Myc-Driven Group 3 Medulloblastoma.

The most aggressive of four medulloblastoma (MB) subgroups are cMyc-driven group 3 (G3) tumors, some of which overexpress EZH2, the histone H3K27 mono-, di-, and trimethylase of polycomb-repressive complex 2. Ezh2 has a context-dependent role in different cancers as an oncogene or tumor suppressor and retards tumor progression in a mouse model of G3 MB. Engineered deletions of Ezh2 in G3 MBs by gene editing nucleases accelerated tumorigenesis, whereas Ezh2 re-expression reversed attendant histone modifications and slowed tumor progression. Candidate oncogenic drivers suppressed by Ezh2 included Gfi1, a proto-oncogene frequently activated in human G3 MBs. Gfi1 disruption antagonized the tumor-promoting effects of Ezh2 loss; conversely, Gfi1 overexpression collaborated with Myc to bypass effects of Trp53 inactivation in driving MB progression in primary cerebellar neuronal progenitors. Although negative regulation of Gfi1 by Ezh2 may restrain MB development, Gfi1 activation can bypass these effects.

Targeting CDK4 and CDK6: From Discovery to Therapy.

UNLABELLED: Biochemical and genetic characterization of D-type cyclins, their cyclin D-dependent kinases (CDK4 and CDK6), and the polypeptide CDK4/6 inhibitor p16(INK4)over two decades ago revealed how mammalian cells regulate entry into the DNA synthetic (S) phase of the cell-division cycle in a retinoblastoma protein-dependent manner. These investigations provided proof-of-principle that CDK4/6 inhibitors, particularly when combined with coinhibition of allied mitogen-dependent signal transduction pathways, might prove valuable in cancer therapy. FDA approval of the CDK4/6 inhibitor palbociclib used with the aromatase inhibitor letrozole for breast cancer treatment highlights long-sought success. The newest findings herald clinical trials targeting other cancers. SIGNIFICANCE: Rapidly emerging data with selective inhibitors of CDK4/6 have validated these cell-cycle kinases as anticancer drug targets, corroborating longstanding preclinical predictions. This review addresses the discovery of these CDKs and their regulators, as well as translation of CDK4/6 biology to positive clinical outcomes and development of rational combinatorial therapies.

Forging a signature of in vivo senescence.

'Cellular senescence', a term originally defining the characteristics of cultured cells that exceed their replicative limit, has been broadened to describe durable states of proliferative arrest induced by disparate stress factors. Proposed relationships between cellular senescence, tumour suppression, loss of tissue regenerative capacity and ageing suffer from lack of uniform definition and consistently applied criteria. Here, we highlight caveats in interpreting the importance of suboptimal senescence-associated biomarkers, expressed either alone or in combination. We advocate that more-specific descriptors be substituted for the now broadly applied umbrella term 'senescence' in defining the suite of diverse physiological responses to cellular stress.

Janus kinase inhibition by ruxolitinib extends dasatinib- and dexamethasone-induced remissions in a mouse model of Ph+ ALL.

Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) is initiated and driven by the oncogenic fusion protein BCR-ABL, a constitutively active tyrosine kinase. Despite major advances in the treatment of this highly aggressive disease with potent inhibitors of the BCR-ABL kinase such as dasatinib, patients in remission frequently relapse due to persistent minimal residual disease possibly supported, at least in part, by salutary cytokine-driven signaling within the hematopoietic microenvironment. Using a mouse model of Ph+ ALL that accurately mimics the genetics, clinical behavior, and therapeutic response of the human disease, we show that a combination of 2 agents approved by the US Food and Drug Administration (dasatinib and ruxolitinib, which inhibit BCR-ABL and Janus kinases, respectively), significantly extends survival by targeting parallel signaling pathways. Although the BCR-ABL kinase cancels the cytokine requirement of immature leukemic B cells, dasatinib therapy restores cytokine dependency and sensitizes leukemic cells to ruxolitinib. As predicted, ruxolitinib alone had no significant antileukemic effect in this model, but it prevented relapse when administered with dasatinib. The combination of dasatinib, ruxolitinib, and the corticosteroid dexamethasone yielded more durable remissions, in some cases after completion of therapy, avoiding the potential toxicity of other cytotoxic chemotherapeutic agents.

CDK9-mediated transcription elongation is required for MYC addiction in hepatocellular carcinoma.

One-year survival rates for newly diagnosed hepatocellular carcinoma (HCC) are <50%, and unresectable HCC carries a dismal prognosis owing to its aggressiveness and the undruggable nature of its main genetic drivers. By screening a custom library of shRNAs directed toward known drug targets in a genetically defined Myc-driven HCC model, we identified cyclin-dependent kinase 9 (Cdk9) as required for disease maintenance. Pharmacological or shRNA-mediated CDK9 inhibition led to robust anti-tumor effects that correlated with MYC expression levels and depended on the role that both CDK9 and MYC exert in transcription elongation. Our results establish CDK9 inhibition as a therapeutic strategy for MYC-overexpressing liver tumors and highlight the relevance of transcription elongation in the addiction of cancer cells to MYC.

Host thiopurine methyltransferase status affects mercaptopurine antileukemic effectiveness in a murine model.

BACKGROUND: Thiopurines are used for many cancers, including acute lymphoblastic leukemia (ALL). Patients with an inherited host defect in thiopurine methyltransferase (TPMT) are at high risk for life-threatening toxicity if treated with conventional dosages, but the impact on antileukemic efficacy is less clear. MATERIALS AND METHODS: We treated thiopurine-sensitive BCR-ABL+Arf-null Tpmt+/+ ALL in Tpmt+/+, +/-, or -/- recipient mice to test the impact of the host polymorphism on antileukemic efficacy. RESULTS: Median survival was similar in untreated mice of different Tpmt genotypes (16-18 days). However, in mice treated with low-dose mercaptopurine (such as tolerated by TPMT-/- patients), the difference in 30-day leukemia-free survival by Tpmt genotype was profound: 5% (±9%) for Tpmt+/+ mice, 47% (±26%) for Tpmt+/- mice, and 85% (±14%) for Tpmt-/- mice (P=5×10), indicating a substantial impact of host Tpmt status on thiopurine effectiveness. Among Tpmt+/+ recipient mice, leukemia-free survival improved with higher doses of mercaptopurine (similar to doses tolerated by wild-type patients) compared with lower doses, and at higher doses was comparable (P=0.6) to the survival of Tpmt-/- mice treated with the lower dose. CONCLUSIONS: These findings support the notion that germline polymorphisms in Tpmt affect not only host tissue toxicity but also antitumor effectiveness.

Simultaneous gene editing by injection of mRNAs encoding transcription activator-like effector nucleases into mouse zygotes.

Injection of transcription activator-like effector nucleases (TALEN) mRNAs into mouse zygotes transferred into foster mothers efficiently generated founder mice with heritable mutations in targeted genes. Immunofluorescence visualization of phosphorylated histone 2A (γH2AX) combined with fluorescence in situ hybridization revealed that TALEN pairs targeting the Agouti locus induced site-directed DNA breaks in zygotes within 6 h of injection, an activity that continued at reduced efficiency in two-cell embryos. TALEN-Agouti mRNAs injected into zygotes of brown FvB × C57BL/6 hybrid mice generated completely black pups, confirming that mutations were induced prior to, and/or early after, cell division. Founder mice, many of which were mosaic, transmitted altered Agouti alleles to F1 pups to yield an allelic series of mutant strains. Although mutations were targeted to "spacer" sequences flanked by TALEN binding sites, larger deletions that extended beyond the TALEN-binding sequences were also detected and were similarly inherited through the germ line. Zygotic coinjection of TALEN mRNAs directed to the Agouti, miR-205, and the Arf tumor suppressor loci yielded pups containing frequent and heritable mutations of two or three genes. Simultaneous gene editing in zygotes affords an efficient approach for producing mice with compound mutant phenotypes, bypassing constraints of conventional mouse knockout technology in embryonic stem cells.

Arf tumor suppressor and miR-205 regulate cell adhesion and formation of extraembryonic endoderm from pluripotent stem cells.

Induction of the Arf tumor suppressor (encoded by the alternate reading frame of the Cdkn2a locus) following oncogene activation engages a p53-dependent transcriptional program that limits the expansion of incipient cancer cells. Although the p19(Arf) protein is not detected in most tissues of fetal or young adult mice, it is physiologically expressed in the fetal yolk sac, a tissue derived from the extraembryonic endoderm (ExEn). Expression of the mouse p19(Arf) protein marks late stages of ExEn differentiation in cultured embryoid bodies (EBs) derived from either embryonic stem cells or induced pluripotent stem cells. Arf inactivation delays differentiation of the ExEn lineage within EBs, but not the formation of other germ cell lineages from pluripotent progenitors. Arf is required for the timely induction of ExEn cells in response to Ras/Erk signaling and, in turn, acts through p53 to ensure the development, but not maintenance, of the ExEn lineage. Remarkably, a significant temporal delay in ExEn differentiation detected during the maturation of Arf-null EBs is rescued by enforced expression of mouse microRNA-205 (miR-205), a microRNA up-regulated by p19(Arf) and p53 that controls ExEn cell migration and adhesion. The noncanonical and canonical roles of Arf in ExEn development and tumor suppression, respectively, may be conceptually linked through mechanisms that govern cell attachment and migration.

Epigenetic regulation of the Ink4a-Arf (Cdkn2a) tumor suppressor locus in the initiation and progression of Notch1-driven T cell acute lymphoblastic leukemia.

Activating mutations of NOTCH1 and deletion of the INK4A-ARF (CDKN2A) tumor suppressor locus are two of the most frequent genetic alterations in T cell acute lymphoblastic leukemia (T-ALL). In a murine model of T-ALL induced by the intracellular domain of Notch1 (ICN1), the genetic interaction between ICN1 signaling and Arf inactivation is developmentally stage-specific, with a more pronounced requirement for Arf deletion in thymocytes than in bone marrow precursors targeted for transformation. In the thymus, the target cell for transformation is a CD4 and CD8 double-negative progenitor that undergoes T cell receptor beta-chain rearrangement, a cell type in which polycomb silencing of Ink4a-Arf is normally requisite. Epigenetic remodeling during tumor progression licenses Arf as a tumor suppressor and in turn provides the selective pressure for Ink4a-Arf deletion in clonal T-ALLs that emerge.

Ink4-Arf locus in cancer and aging.

Three tumor suppressor genes at the small (<50 kb) INK4-ARF (CDKN2A/B) locus on human chromosome 9p21 coordinate a signaling network that depends on the activities of the retinoblastoma (RB) protein and the p53 transcription factor. Disruption of this circuitry, frequently by codeletion of INK4-ARF, is a hallmark of cancer, begging the question of why the intimate genetic linkage of these tumor suppressor genes has been maintained in mammals despite the risk of their coinactivation. The INK4-ARF locus is not highly expressed under normal physiologic conditions in young mammals, but its induction becomes more pronounced as animals age. Notably, INK4-ARF is actively silenced en bloc in embryonic, fetal, and adult stem cells but becomes poised to respond to oncogenic stress signals as stem cells lose their self-renewal capacity and differentiate, thereby providing a potent barrier to tumor formation. Epigenetic remodeling of the locus as a whole provides a mechanism for coordinating the activities of RB and p53. A hypothesis is that the INK4-ARF locus may have evolved to physiologically restrict the self-renewal capacities and numbers of stem and progenitor cells with the attendant consequence of limiting tissue regenerative capacity, particularly as animals age. Deletion of INK4-ARF contributes to the aberrant self-renewal capacity of tumor cells and occurs frequently in many forms of human cancer.