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.

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.

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.

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.

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.

Divorcing ARF and p53: an unsettled case.

Mammalian cells that sustain oncogenic insults can invoke defensive programmes that either halt their division or trigger their apoptosis, but these countermeasures must be finely tuned to discriminate between physiological and potentially harmful growth-promoting states. By functioning specifically to oppose abnormally prolonged and sustained proliferative signals produced by activated oncogenes, the ARF tumour suppressor antagonizes functions of MDM2 to induce protective responses that depend on the p53 transcription factor and its many target genes. However, ARF has been reported to physically associate with proteins other than MDM2 and to have p53-independent activities, most of which remain controversial and poorly understood.

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.

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. Instead, inactivation of Arf triggers germ cell-autonomous, p53-dependent apoptosis of primary spermatocytes in late meiotic prophase, resulting in reduced sperm production. Arf deficiency also causes premature, elevated, and persistent accumulation of the phosphorylated histone variant H2AX, reduces numbers of chromosome-associated complexes of Rad51 and Dmc1 recombinases during meiotic prophase, and yields incompletely synapsed autosomes during pachynema. Inactivation of Ink4a increases the fraction of spermatogonia in S-phase and restores sperm numbers in Ink4a-Arf doubly deficient mice but does not abrogate γ-H2AX accumulation in spermatocytes or p53-dependent apoptosis resulting from Arf inactivation. Thus, as opposed to its canonical role as a tumor suppressor in inducing p53-dependent senescence or apoptosis, Arf expression in spermatogonia instead initiates a salutary feed-forward program that prevents p53-dependent apoptosis, contributing to the survival of meiotic male germ cells.

Rescue of key features of the p63-null epithelial phenotype by inactivation of Ink4a and Arf.

Mice lacking p63 cannot form skin, exhibit craniofacial and skeletal defects, and die soon after birth. The p63 gene regulates a complex network of target genes, and disruption of p63 has been shown to affect the maintenance of epithelial stem cells, the differentiation of keratinocytes, and the preservation of the adhesive properties of stratified epithelium. Here, we show that inactivation of p63 in mice is accompanied by aberrantly increased expression of the Ink4a and Arf tumour suppressor genes. In turn, anomalies of the p63-null mouse affecting the skin and skeleton are partially ameliorated in mice lacking either Ink4a or Arf. Rescue of epithelialization is accompanied by restoration of keratinocyte proliferative capacity both in vivo and in vitro and by expression of markers of squamous differentiation. Thus, in the absence of p63, abnormal upregulation of Ink4a and Arf is incompatible with skin development.

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. Inactivation of the Arf tumor suppressor further increased the self-renewal capacity of the primitive, preleukemic thymocyte pool and accelerated the development of aggressive, Lmo2-induced T-cell lympholeukemias. Notch mutations were frequently detected in these Lmo2-induced tumors. The Arf promoter was not directly engaged by Lmo2 or mutant Notch, and use of a mouse model in which activation of a mutant Notch allele depends on previous engagement of the Arf promoter revealed that Notch activation could occur as a subsequent event in T-cell tumorigenesis. Therefore, Lmo2 cooperates with Arf loss to enhance self-renewal in primitive thymocytes. Notch mutation and Arf inactivation appear to independently cooperate in no requisite order with Lmo2 overexpression in inducing T-ALL, and all 3 events remained insufficient to guarantee immediate tumor development.

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. Although relapse was frequently accompanied by the eventual appearance of leukemic clones harboring BCR-ABL kinase domain (KD) mutations that confer drug resistance, their clonal emergence required prolonged dasatinib exposure. KD P-loop mutations predominated in mice receiving less intensive therapy, whereas high-dose treatment selected for T315I "gatekeeper" mutations resistant to all 3 Food and Drug Administration-approved BCR-ABL kinase inhibitors. The addition of dexamethasone and/or L-asparaginase to reduced-intensity dasatinib therapy improved long-term survival of the majority of mice that received all 3 drugs. Although non-tumor-cell-autonomous mechanisms can prevent full eradication of dasatinib-refractory ALL in this clinically relevant model, the emergence of resistance to BCR-ABL kinase inhibitors can be effectively circumvented by the addition of "conventional" chemotherapeutic agents with alternate antileukemic mechanisms of action.

Hzf regulates adipogenesis through translational control of C/EBPalpha.

Adipocyte differentiation requires a well-defined programme of gene expression in which the transcription factor C/EBPalpha (CCAAT/enhancer-binding protein) has a central function. Here, we show that Hzf (haematopoietic zinc-finger), a previously identified p53 transcriptional target, regulates C/EBPalpha expression. Hzf is induced during differentiation of preadipocyte cell lines, and its suppression by short hairpin RNA disrupts adipogenesis. In Hzf's absence, expression of C/EBPalpha is severely impaired because of reduced translation of its mRNA. Hzf physically interacts with the 3' untranslated region of C/EBPalpha mRNA to enhance its translation. Taken together, these findings underscore a critical role of Hzf in the adipogenesis regulatory cascade.

The RB and p53 pathways in cancer.

The life history of cancer cells encompasses a series of genetic missteps in which normal cells are progressively transformed into tumor cells that invade surrounding tissues and become malignant. Most prominent among the regulators disrupted in cancer cells are two tumor suppressors, the retinoblastoma protein (RB) and the p53 transcription factor. Here, we discuss interconnecting signaling pathways controlled by RB and p53, attempting to explain their potentially universal involvement in the etiology of cancer. Pinpointing the various ways by which the functions of RB and p53 are subverted in individual tumors should provide a rational basis for developing more refined tumor-specific therapies.

Antagonism of Myc functions by Arf.

The Arf-Mdm2-p53 tumor suppressor pathway is activated by sustained hyperproliferative signals emanating from oncoproteins such as Myc. A recent study reveals a novel level of feedback control, whereby induced p19(Arf) binds to Myc and blocks cell proliferation by selectively impairing its transactivation functions.

Transient expression of the Arf tumor suppressor during male germ cell and eye development in Arf-Cre reporter mice.

The Arf tumor suppressor is expressed transiently during mouse male germ cell and eye development. Its inactivation compromises spermatogenesis as mice age and leads to aberrant postnatal proliferation of cells in the vitreous of the eye, resulting in blindness. In the testis, expression of p19(Arf) is limited to spermatogonia but is extinguished completely in spermatocytes, suggesting that Arf plays a physiologic role in setting the balance between mitotic and meiotic germ cell division. A knock-in allele encoding Cre recombinase regulated by the mouse cellular Arf promoter was used to trace Arf gene induction in vivo. Interbreeding to a reporter strain that expresses Cre-dependent YFP provided proof-of-principle that the Arf-Cre allele was appropriately expressed in the male germ cell lineage. However, Cre expression resulted in male sterility, limiting germ line transmission of the knock-in allele to females. Arf-null mice fail to resorb the hyaloid vasculature within the ocular vitreous where pericyte-like cells that express the PDGF-beta receptor (Pdgfrbeta) proliferate aberrantly and destroy the retina and lens. Interbreeding of Arf-Cre females to males containing "floxed" (FL) Arf alleles yielded Arf(Cre/FL) progeny that exhibited variably penetrant defects in visual acuity ranging to total blindness. Crossing the Arf(Cre/FL) alleles onto a Pdgfrbeta(FL/FL) background normalized all histopathology and restored vision fully.

Stage-specific Arf tumor suppression in Notch1-induced T-cell acute lymphoblastic leukemia.

Frequent hallmarks of T-cell acute lymphoblastic leukemia (T-ALL) include aberrant NOTCH signaling and deletion of the CDKN2A locus, which contains 2 closely linked tumor suppressor genes (INK4A and ARF). When bone marrow cells or thymocytes transduced with a vector encoding the constitutively activated intracellular domain of Notch1 (ICN1) are expanded ex vivo under conditions that support T-cell development, cultured progenitors rapidly induce CD4+/CD8+ T-ALLs after infusion into healthy syngeneic mice. Under these conditions, enforced ICN1 expression also drives formation of T-ALLs in unconditioned CD-1 nude mice, bypassing any requirements for thymic maturation. Retention of Arf had relatively modest activity in suppressing the formation of T-ALLs arising from bone marrow-derived ICN1+ progenitors in which the locus is epigenetically silenced, and all resulting Arf (+/+) tumors failed to express the p19(Arf) protein. In striking contrast, retention of Arf in thymocyte-derived ICN1+ donor cells significantly delayed disease onset and suppressed the penetrance of T-ALL. Use of cultured thymocyte-derived donor cells expressing a functionally null Arf-GFP knock-in allele confirmed that ICN1 signaling can induce Arf expression in vivo. Arf activation by ICN1 in T cells thereby provides stage-specific tumor suppression but also a strong selective pressure for deletion of the locus in T-ALL.

Two tumor suppressors, p27Kip1 and patched-1, collaborate to prevent medulloblastoma.

Two cyclin-dependent kinase inhibitors, p18(Ink4c) and p27(Kip1), are required for proper cerebellar development. Loss of either of these proteins conferred a proliferative advantage to granule neuron progenitors, although inactivation of Kip1 exerted a greater effect. Mice heterozygous for Patched-1 (Ptc1+/-) that are either heterozygous or nullizygous for Kip1 developed medulloblastoma rapidly and with high penetrance. All tumors from Ptc1+/-;Kip1+/- or Ptc1+/-;Kip1-/- mice failed to express the wild-type Ptc1 allele, consistent with its role as a canonical "two-hit" tumor suppressor. In contrast, expression of the wild-type p27(Kip1) protein was invariably maintained in medulloblastomas arising in Ptc1+/-;Kip1+/- mice, indicating that Kip1 is haploinsufficient for tumor suppression. Although medulloblastomas occurring in Ptc1+/- mice were histopathologically heterogeneous and contained intermixed regions of both rapidly proliferating and nondividing more differentiated cells, tumors that also lacked Kip1 were uniformly less differentiated, more highly proliferative, and invasive. Molecular analysis showed that the latter medulloblastomas exhibited constitutive activation of the Sonic hedgehog signaling pathway without loss of functional p53. Apart from gains or losses of single chromosomes, with gain of chromosome 6 being the most frequent, no other chromosomal anomalies were identified by spectral karyotyping, and half of the medulloblastomas so examined retained a normal karyotype. In this respect, this mouse medulloblastoma model recapitulates the vast majority of human medulloblastomas that do not sustain TP53 mutations and are not aneuploid.

Hzf, a p53-responsive gene, regulates maintenance of the G2 phase checkpoint induced by DNA damage.

The hematopoietic zinc finger protein, Hzf, is induced in response to genotoxic and oncogenic stress. The Hzf protein is encoded by a p53-responsive gene, and its overexpression, either in cells retaining or lacking functional 53, halts their proliferation. Enforced expression of Hzf led to the appearance of tetraploid cells with supernumerary centrosomes and, ultimately, to cell death. Eliminating Hzf mRNA expression by use of short hairpin (sh) RNAs had no overt effect on unstressed cells but inhibited the maintenance of G2 phase arrest following ionizing radiation (IR), thereby sensitizing cells to DNA damage. Canonical p53-responsive gene products such as p21Cip1 and Mdm2 were induced by IR in cells treated with Hzf shRNA. However, the reduction in the level of Hzf protein was accompanied by increased polyubiquitination and turnover of p21Cip1, an inhibitor of cyclin-dependent kinases whose expression contributes to maintaining the duration of the G2 checkpoint in cells that have sustained DNA damage. Thus, two p53-inducible gene products, Hzf and p21Cip1, act concomitantly to enforce the G(2) checkpoint.