PARP-1 selectively impairs KRAS-driven phenotypic and molecular features in intrahepatic cholangiocarcinoma.

OBJECTIVE: Intrahepatic cholangiocarcinoma (iCCA) is the second most common primary liver cancer with limited therapeutic options. KRAS mutations are among the most abundant genetic alterations in iCCA associated with poor clinical outcome and treatment response. Recent findings indicate that Poly(ADP-ribose)polymerase1 (PARP-1) is implicated in KRAS-driven cancers, but its exact role in cholangiocarcinogenesis remains undefined. DESIGN: PARP-1 inhibition was performed in patient-derived and established iCCA cells using RNAi, CRISPR/Cas9 and pharmacological inhibition in KRAS-mutant, non-mutant cells. In addition, Parp-1 knockout mice were combined with iCCA induction by hydrodynamic tail vein injection to evaluate an impact on phenotypic and molecular features of Kras-driven and Kras-wildtype iCCA. Clinical implications were confirmed in authentic human iCCA. RESULTS: PARP-1 was significantly enhanced in KRAS-mutant human iCCA. PARP-1-based interventions preferentially impaired cell viability and tumourigenicity in human KRAS-mutant cell lines. Consistently, loss of Parp-1 provoked distinct phenotype in Kras/Tp53-induced versus Akt/Nicd-induced iCCA and abolished Kras-dependent cholangiocarcinogenesis. Transcriptome analyses confirmed preferential impairment of DNA damage response pathways and replicative stress response mediated by CHK1. Consistently, inhibition of CHK1 effectively reversed PARP-1 mediated effects. Finally, Parp-1 depletion induced molecular switch of KRAS-mutant iCCA recapitulating good prognostic human iCCA patients. CONCLUSION: Our findings identify the novel prognostic and therapeutic role of PARP-1 in iCCA patients with activation of oncogenic KRAS signalling.

Multi-omic and functional analysis for classification and treatment of sarcomas with FUS-TFCP2 or EWSR1-TFCP2 fusions.

Linking clinical multi-omics with mechanistic studies may improve the understanding of rare cancers. We leverage two precision oncology programs to investigate rhabdomyosarcoma with FUS/EWSR1-TFCP2 fusions, an orphan malignancy without effective therapies. All tumors exhibit outlier ALK expression, partly accompanied by intragenic deletions and aberrant splicing resulting in ALK variants that are oncogenic and sensitive to ALK inhibitors. Additionally, recurrent CKDN2A/MTAP co-deletions provide a rationale for PRMT5-targeted therapies. Functional studies show that FUS-TFCP2 blocks myogenic differentiation, induces transcription of ALK and truncated TERT, and inhibits DNA repair. Unlike other fusion-driven sarcomas, TFCP2-rearranged tumors exhibit genomic instability and signs of defective homologous recombination. DNA methylation profiling demonstrates a close relationship with undifferentiated sarcomas. In two patients, sarcoma was preceded by benign lesions carrying FUS-TFCP2, indicating stepwise sarcomagenesis. This study illustrates the potential of linking precision oncology with preclinical research to gain insight into the classification, pathogenesis, and therapeutic vulnerabilities of rare cancers.

Transcriptional Response to Standard AML Drugs Identifies Synergistic Combinations.

Unlike genomic alterations, gene expression profiles have not been widely used to refine cancer therapies. We analyzed transcriptional changes in acute myeloid leukemia (AML) cell lines in response to standard first-line AML drugs cytarabine and daunorubicin by means of RNA sequencing. Those changes were highly cell- and treatment-specific. By comparing the changes unique to treatment-sensitive and treatment-resistant AML cells, we enriched for treatment-relevant genes. Those genes were associated with drug response-specific pathways, including calcium ion-dependent exocytosis and chromatin remodeling. Pharmacological mimicking of those changes using EGFR and MEK inhibitors enhanced the response to daunorubicin with minimum standalone cytotoxicity. The synergistic response was observed even in the cell lines beyond those used for the discovery, including a primary AML sample. Additionally, publicly available cytotoxicity data confirmed the synergistic effect of EGFR inhibitors in combination with daunorubicin in all 60 investigated cancer cell lines. In conclusion, we demonstrate the utility of treatment-evoked gene expression changes to formulate rational drug combinations. This approach could improve the standard AML therapy, especially in older patients.

Synergistic targeting of FLT3 mutations in AML via combined menin-MLL and FLT3 inhibition.

The interaction of menin (MEN1) and MLL (MLL1, KMT2A) is a dependency and provides a potential opportunity for treatment of NPM1-mutant (NPM1mut) and MLL-rearranged (MLL-r) leukemias. Concomitant activating driver mutations in the gene encoding the tyrosine kinase FLT3 occur in both leukemias and are particularly common in the NPM1mut subtype. In this study, transcriptional profiling after pharmacological inhibition of the menin-MLL complex revealed specific changes in gene expression, with downregulation of the MEIS1 transcription factor and its transcriptional target gene FLT3 being the most pronounced. Combining menin-MLL inhibition with specific small-molecule kinase inhibitors of FLT3 phosphorylation resulted in a significantly superior reduction of phosphorylated FLT3 and transcriptional suppression of genes downstream of FLT3 signaling. The drug combination induced synergistic inhibition of proliferation, as well as enhanced apoptosis, compared with single-drug treatment in models of human and murine NPM1mut and MLL-r leukemias harboring an FLT3 mutation. Primary acute myeloid leukemia (AML) cells harvested from patients with NPM1mutFLT3mut AML showed significantly better responses to combined menin and FLT3 inhibition than to single-drug or vehicle control treatment, whereas AML cells with wild-type NPM1, MLL, and FLT3 were not affected by either of the 2 drugs. In vivo treatment of leukemic animals with MLL-r FLT3mut leukemia reduced leukemia burden significantly and prolonged survival compared with results in the single-drug and vehicle control groups. Our data suggest that combined menin-MLL and FLT3 inhibition represents a novel and promising therapeutic strategy for patients with NPM1mut or MLL-r leukemia and concurrent FLT3 mutation.

SIRT1 prevents genotoxic stress-induced p53 activation in acute myeloid leukemia.

SIRT1 is an important regulator of cellular stress response and genomic integrity. Its role in tumorigenesis is controversial. Whereas sirtuin 1 (SIRT1) can act as a tumor suppressor in some solid tumors, increased expression has been demonstrated in many cancers, including hematologic malignancies. In chronic myeloid leukemia, SIRT1 promoted leukemia development, and targeting SIRT1 sensitized chronic myeloid leukemia progenitors to tyrosine kinase inhibitor treatment. In this study, we investigated the role of SIRT1 in acute myeloid leukemia (AML). We show that SIRT1 protein, but not RNA levels, is overexpressed in AML samples harboring activating mutations in signaling pathways. In FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD)(+)-cells protein, expression of SIRT1 is regulated by FLT3 kinase activity. In addition, SIRT1 function is modulated via the ATM-DBC1-SIRT1 axis in a FLT3-ITD-dependent manner. In murine leukemia models driven by MLL-AF9 or AML1-ETO coexpressing FLT3-ITD, SIRT1 acts as a safeguard to counteract oncogene-induced stress, and leukemic blasts become dependent on SIRT1 activity. Pharmacologic targeting or RNAi-mediated knockdown of SIRT1 inhibited cell growth and sensitized AML cells to tyrosine kinase inhibitor treatment and chemotherapy. This effect was a result of the restoration of p53 activity. Our data suggest that targeting SIRT1 represents an attractive therapeutic strategy to overcome primary resistance in defined subsets of patients with AML.

Targeting components of the alternative NHEJ pathway sensitizes KRAS mutant leukemic cells to chemotherapy.

Activating KRAS mutations are detected in a substantial number of hematologic malignancies. In a murine T-cell acute lymphoblastic leukemia (T-ALL) model, we previously showed that expression of oncogenic Kras induced a premalignant state accompanied with an arrest in T-cell differentiation and acquisition of somatic Notch1 mutations. These findings prompted us to investigate whether the expression of oncogenic KRAS directly affects DNA damage repair. Applying divergent, but complementary, genetic approaches, we demonstrate that the expression of KRAS mutants is associated with increased expression of DNA ligase 3α, poly(ADP-ribose) polymerase 1 (PARP1), and X-ray repair cross-complementing protein 1 (XRCC1), all essential components of the error-prone, alternative nonhomologous end-joining (alt-NHEJ) pathway. Functional studies revealed delayed repair kinetics, increased misrepair of DNA double-strand breaks, and the preferential use of microhomologous DNA sequences for end joining. Similar effects were observed in primary murine T-ALL blasts. We further show that KRAS-mutated cells, but not KRAS wild-type cells, rely on the alt-NHEJ repair pathway on genotoxic stress. RNA interference-mediated knockdown of DNA ligase 3α abolished resistance to apoptotic cell death in KRAS-mutated cells. Our data indicate that targeting components of the alt-NHEJ pathway sensitizes KRAS-mutated leukemic cells to standard chemotherapeutics and represents a promising approach for inducing synthetic lethal vulnerability in cells harboring otherwise nondruggable KRAS mutations.

STAT5 is crucial to maintain leukemic stem cells in acute myelogenous leukemias induced by MOZ-TIF2.

MOZ-TIF2 is a leukemogenic fusion oncoprotein that confers self-renewal capability to hematopoietic progenitor cells and induces acute myelogenous leukemia (AML) with long latency in bone marrow transplantation assays. Here, we report that FLT3-ITD transforms hematopoietic cells in cooperation with MOZ-TIF2 in vitro and in vivo. Coexpression of FLT3-ITD confers growth factor independent survival/proliferation, shortens disease latency, and results in an increase in the number of leukemic stem cells (LSC). We show that STAT5, a major effector of aberrant FLT3-ITD signal transduction, is both necessary and sufficient for this cooperative effect. In addition, STAT5 signaling is essential for MOZ-TIF2-induced leukemic transformation itself. Lack of STAT5 in fetal liver cells caused rapid differentiation and loss of replating capacity of MOZ-TIF2-transduced cells enriched for LSCs. Furthermore, mice serially transplanted with Stat5(-/-) MOZ-TIF2 leukemic cells develop AML with longer disease latency and finally incomplete penetrance when compared with mice transplanted with Stat5(+/+) MOZ-TIF2 leukemic cells. These data suggest that STAT5AB is required for the self-renewal of LSCs and represents a combined signaling node of FLT3-ITD and MOZ-TIF2 driven leukemogenesis. Therefore, targeting aberrantly activated STAT5 or rewired downstream signaling pathways may be a promising therapeutic option.

Pharmacologic inhibition of mTOR antagonizes the cytotoxic activity of pemetrexed in non-small cell lung cancer.

PURPOSE: Pemetrexed, an inhibitor of thymidylate synthase (TS) and additional folate-dependent enzymes, is clinically active in patients suffering from "non-squamous" non-small cell lung cancer (NSCLC). High expression of TS has been implied as biomarker predictive of resistance to pemetrexed. Against this background, we studied whether inhibition of mTOR could lower expression of TS and thus sensitize NSCLC cells to pemetrexed. METHODS AND RESULTS: Using squamous cell carcinoma and adenocarcinoma NSCLC cell lines, we observed that constitutive TS expression levels failed to correlate with sensitivity to growth inhibition or apoptosis imposed by pemetrexed in vitro. Interestingly, pemetrexed strongly induced TS RNA and protein expression in all cell lines. The allosteric "rapalogue" mTOR inhibitor everolimus suppressed constitutive, but not pemetrexed-induced TS expression. Surprisingly, cotreatment with everolimus protected NSCLC cells against pemetrexed-induced apoptosis. This resulted in increased long-term clonogenic survival of NSCLC cells treated with pemetrexed plus everolimus as compared to pemetrexed alone. No such negative interaction was observed when everolimus was combined with recombinant TRAIL, a proliferation-independent proapoptotic agent. CONCLUSIONS: Rapalogues may suppress the antitumor activity of pemetrexed by slowing cell cycle progression. This should be considered when combining pemetrexed and mTOR inhibitors in NSCLC treatment.

Functional and clinical characterization of the putative tumor suppressor WWOX in non-small cell lung cancer.

INTRODUCTION: The oxidoreductase WWOX was initially described as a putative tumor suppressor in breast cancer. Non-small cell lung cancers (NSCLCs) frequently show aberrant WWOX expression. Herein, we characterized WWOX at a functional level in preclinical NSCLC models and in primary NSCLC biopsies. METHODS: The human wild-type (wt) WWOX complementary DNA and a mutant WWOX with structurally disrupted short-chain dehydrogenase/reductase domain were conditionally expressed at physiological levels in several human NSCLC models. Resulting transgenic cell populations were analyzed with respect to clonogenic survival and apoptosis sensitivity in vitro and tumor growth in immune-deficient mice. Tissue microarrays prepared from surgically resected primary human NSCLC tumors were studied to correlate intratumoral WWOX expression with patient outcomes. RESULTS: Conditional expression of wt WWOX, but not mutant WWOX, suppressed clonogenic survival of NSCLC cells in vitro and tumor growth in vivo. In addition, preserved intratumoral WWOX expression was associated with improved outcome in a cohort of 85 patients with surgically resected NSCLC. Unexpectedly, wt WWOX failed to sensitize NSCLC cells to various apoptotic stimuli but robustly protected against apoptosis induced by inhibitors of growth factor signal transduction. CONCLUSIONS: WWOX acts as a tumor suppressor in human NSCLC models in a short-chain dehydrogenase/reductase domain-dependent manner. This activity is independent of sensitization to apoptotic cell death. WWOX expression as detected by immunohistochemistry may be a prognostic biomarker in surgically resected, early-stage NSCLC.

Perforin deficiency attenuates inflammation and tumor growth in colitis-associated cancer.

BACKGROUND: Patients with inflammatory bowel disease (IBD) have a markedly increased risk to develop colon cancer, but there are only limited data about the host antitumor response in such colitis-associated cancer. In the present study we aimed at assessing the role of perforin-dependent effector mechanisms in the immune response in a murine model of colitis-associated colon cancer. METHODS: Wildtype and perforin-deficient mice were analyzed in a mouse model of colitis-associated colon cancer using azoxymethane (AOM) and dextran sodium sulfate (DSS). RESULTS: Tumors of wildtype mice showed infiltration of CD4+, CD8+ T cells, natural killer (NK) cells, high numbers of apoptotic cells, and expression of the transcription factor eomesodermin and cytotoxic effector proteins, suggesting a potential role of the antitumor immune response in AOM/DSS tumorigenesis. Furthermore, perforin deficiency resulted in reduced apoptosis of epithelial cells as compared to wildtype mice, whereas tumor infiltration by NK cells, CD8+, and CD4+ T cells was unchanged. However, perforin-deficient mice surprisingly developed significantly fewer tumors than wildtype mice. Subsequent studies identified an important role of perforin in regulating colitis activity, as perforin deficiency caused a significant reduction of DSS colitis activity and proinflammatory cytokine production as compared to wildtype controls. CONCLUSIONS: Perforin is involved in both the antitumor immune response and the regulation of activity of mucosal inflammation in colitis-associated cancer. Our data emphasize the possible consequences for therapeutic strategies targeting colitis-associated colon cancer.

RASSF1A mediates p21Cip1/Waf1-dependent cell cycle arrest and senescence through modulation of the Raf-MEK-ERK pathway and inhibition of Akt.

Promoter hypermethylation preventing expression of the RAS association domain family 1 isoform A (RASSF1A) gene product is among the most abundant epigenetic deregulations in human cancer. Restoration of RASSF1A inhibits tumor cell growth in vitro and in murine xenograft models. Rassf1a-deficient mice feature increased spontaneous and carcinogen-induced tumor formation. Mechanistically, RASSF1A affects several cellular functions, such as microtubule dynamics, migration, proliferation, and apoptosis; however, its tumor-suppressive mechanism is incompletely understood. To study the functional consequences of RASSF1A expression in human cancer cells, we made use of a doxycycline-inducible expression system and a RASSF1A-deficient lung cancer cell line. We observed that RASSF1A induces cell cycle arrest in G(1) phase and senescence in vitro and in tumors established in immunodeficient mice. RASSF1A-mediated growth inhibition was accompanied by the up-regulation of the cyclin-dependent kinase inhibitor p21(Cip1/Waf1) and proceeded independently of p53, p14(Arf), and p16(Ink4a). Loss of p21(Cip1/Waf1) or coexpression of the human papilloma virus 16 oncoprotein E7 was found to override RASSF1A-induced cell cycle arrest and senescence. Conditional RASSF1A affected mitogen-activated protein kinase and protein kinase B/Akt signaling to up-regulate p21(Cip1/Waf1) and to facilitate its nuclear localization. In summary, RASSF1A can mediate cell cycle arrest and senescence in human cancer cells by p53-independent regulation of p21(Cip1/Waf1).

Targeting Bcl-2 family proteins modulates the sensitivity of B-cell lymphoma to rituximab-induced apoptosis.

The chimeric monoclonal antibody rituximab is the standard of care for patients with B-cell non-Hodgkin lymphoma (B-NHL). Rituximab mediates complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity of CD20-positive human B cells. In addition, rituximab sensitizes B-NHL cells to cytotoxic chemotherapy and has direct apoptotic and antiproliferative effects. Whereas expression of the CD20 antigen is a natural prerequisite for rituximab sensitivity, cell-autonomous factors determining the response of B-NHL to rituximab are less defined. To this end, we have studied rituximab-induced apoptosis in human B-NHL models. We find that rituximab directly triggers apoptosis via the mitochondrial pathway of caspase activation. Expression of antiapoptotic Bcl-xL confers resistance against rituximab-induced apoptosis in vitro and rituximab treatment of xenografted B-NHL in vivo. B-NHL cells insensitive to rituximab-induced apoptosis exhibit increased endogenous expression of multiple antiapoptotic Bcl-2 family proteins, or activation of phosphatidylinositol-3-kinase signaling resulting in up-regulation of Mcl-1. The former resistance pattern is overcome by treatment with the BH3-mimetic ABT-737, the latter by combining rituximab with pharmacologic phosphatidylinositol-3-kinase inhibitors. In conclusion, sensitivity of B-NHL cells to rituximab-induced apoptosis is determined at the level of mitochondria. Pharmacologic modulation of Bcl-2 family proteins or their upstream regulators is a promising strategy to overcome rituximab resistance.

Targeting AKT signaling sensitizes cancer to cellular immunotherapy.

The promise of cancer immunotherapy is long-term disease control with high specificity and low toxicity. However, many cancers fail immune interventions, and secretion of immunosuppressive factors, defective antigen presentation, and expression of death ligands or serpins are regarded as main escape mechanisms. Here, we study whether deregulation of growth and survival factor signaling, which is encountered in most human cancers, provides another level of protection against immunologic tumor eradication. We show in two models that activated cell autonomous protein kinase B (PKB)/AKT signaling mediates resistance against tumor suppression by antigen-specific CTLs in vitro and adoptively transferred cellular immune effectors in vivo. PKB/AKT-dependent immunoresistance of established tumors is reversed by genetic suppression of endogenous Mcl-1, an antiapoptotic member of the Bcl-2 family. Mechanistically, deregulated PKB/AKT stabilizes Mcl-1 expression in a mammalian target of rapamycin (mTOR)-dependent pathway. Treatment with the mTOR inhibitor rapamycin effectively sensitizes established cancers to adoptive immunotherapy in vivo. In conclusion, cancer cell-intrinsic PKB/AKT signaling regulates the susceptibility to immune-mediated cytotoxicity. Combined targeting of signal transduction pathways may be critical for improvement of cancer immunotherapies.

An immune escape screen reveals Cdc42 as regulator of cancer susceptibility to lymphocyte-mediated tumor suppression.

Adoptive cellular immunotherapy inducing a graft-versus-tumor (GVT) effect is the therapeutic mainstay of allogeneic hematopoietic stem cell transplantation (ASCT) for high-risk leukemias. Autologous immunotherapies using vaccines or adoptive transfer of ex vivo-manipulated lymphocytes are clinically explored in patients with various cancer entities. Main reason for failure of ASCT and cancer immunotherapy is progression of the underlying malignancy, which is more prevalent in patients with advanced disease. Elucidating the molecular mechanisms contributing to immune escape will help to develop strategies for the improvement of immunologic cancer treatment. To this end, we have undertaken functional screening and expression cloning of factors mediating resistance to antigen-specific cytotoxic T lymphocytes (CTLs). We have identified Cdc42, a GTPase regulating actin dynamics and growth factor signaling that is highly expressed in invasive cancers, as determinator of cancer cell susceptibility to antigen-specific CTLs in vitro and adoptively transferred immune effectors in vivo. Cdc42 prevents CTL-induced apoptosis via mitogen-activated protein kinase (MAPK) signaling and posttranscriptional stabilization of Bcl-2. Pharmacologic inhibition of MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK) overcomes Cdc42-mediated immunoresistance and activation of Bcl-2 in vivo. In conclusion, Cdc42 signaling contributes to immune escape of cancer. Targeting Cdc42 may improve the efficacy of cancer immunotherapies.