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.

Sexually dimorphic tumor suppression by small mitochondrial .

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).

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.

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

The mouse p19 (human p14) tumor suppressor protein, encoded in part from an alternative reading frame of the () gene, inhibits the Mdm2 E3 ubiquitin ligase to activate p53. 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 mRNA encodes two distinct polypeptides, including full-length p19 and N-terminally truncated and unstable p15 ("small mitochondrial Arf") initiated from an internal in-frame AUG codon specifying methionine-45.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Expression of arf tumor suppressor in spermatogonia facilitates meiotic progression in male germ cells.

The mammalian Cdkn2a (Ink4a-Arf) locus encodes two tumor suppressor proteins (p16(Ink4a) and p19(Arf)) that respectively enforce the anti-proliferative functions of the retinoblastoma protein (Rb) and the p53 transcription factor in response to oncogenic stress. Although p19(Arf) is not normally detected in tissues of young adult mice, a notable exception occurs in the male germ line, where Arf is expressed in spermatogonia, but not in meiotic spermatocytes arising from them. Unlike other contexts in which the induction of Arf potently inhibits cell proliferation, expression of p19(Arf) in spermatogonia does not interfere with mitotic cell division.

Functional interactions between Lmo2, the Arf tumor suppressor, and Notch1 in murine T-cell malignancies.

LMO2 is a target of chromosomal translocations in T-cell tumors and was activated by retroviral vector insertions in T-cell tumors from X-SCID patients in gene therapy trials. To better understand the cooperating genetic events in LMO2-associated T-cell acute lymphoblastic leukemia (T-ALL), we investigated the roles of Arf tumor suppressor loss and Notch activation in murine models of transplantation. Lmo2 overexpression enhanced the expansion of primitive DN2 thymocytes, eventually facilitating the stochastic induction of clonal CD4(+)/CD8(+) malignancies.

Chemotherapeutic agents circumvent emergence of dasatinib-resistant BCR-ABL kinase mutations in a precise mouse model of Philadelphia chromosome-positive acute lymphoblastic leukemia.

The introduction of cultured p185(BCR-ABL)-expressing (p185+) Arf (-/-) pre-B cells into healthy syngeneic mice induces aggressive acute lymphoblastic leukemia (ALL) that genetically and phenotypically mimics the human disease. We adapted this high-throughput Philadelphia chromosome-positive (Ph(+)) ALL animal model for in vivo luminescent imaging to investigate disease progression, targeted therapeutic response, and ALL relapse in living mice. Mice bearing high leukemic burdens (simulating human Ph(+) ALL at diagnosis) entered remission on maximally intensive, twice-daily dasatinib therapy, but invariably relapsed with disseminated and/or central nervous system disease.

Atoh1 inhibits neuronal differentiation and collaborates with Gli1 to generate medulloblastoma-initiating cells.

The morphogen and mitogen Sonic Hedgehog (Shh) activates a Gli1-dependent transcription program that drives proliferation of granule neuron progenitors (GNP) within the external germinal layer of the postnatally developing cerebellum. Medulloblastomas with mutations activating the Shh signaling pathway preferentially arise within the external germinal layer, and the tumor cells closely resemble GNPs. Atoh1/Math1, a basic helix-loop-helix transcription factor essential for GNP histogenesis, does not induce medulloblastomas when expressed in primary mouse GNPs that are explanted from the early postnatal cerebellum and transplanted back into the brains of naïve mice.