Daniela Bossi

Cell-cycle restriction limits DNA damage and maintains self-renewal of leukaemia stem cells.

Rare cells with the properties of stem cells are integral to the development and perpetuation of leukaemias. A defining characteristic of stem cells is their capacity to self-renew, which is markedly extended in leukaemia stem cells. The underlying molecular mechanisms, however, are largely unknown. Here we demonstrate that expression of the cell-cycle inhibitor p21 is indispensable for maintaining self-renewal of leukaemia stem cells. Expression of leukaemia-associated oncogenes in mouse haematopoietic stem cells (HSCs) induces DNA damage and activates a p21-dependent cellular response, which leads to reversible cell-cycle arrest and DNA repair. Activated p21 is critical in preventing excess DNA-damage accumulation and functional exhaustion of leukaemic stem cells. These data unravel the oncogenic potential of p21 and suggest that inhibition of DNA repair mechanisms might function as potent strategy for the eradication of the slowly proliferating leukaemia stem cells.

The methyl-CpG binding protein MBD1 is required for PML-RARα function

PML-RARα induces a block of hematopoietic differentiation and acute promyelocytic leukemia. This block is based on its capacity to inactivate target genes by recruiting histone deacetylase (HDAC) and DNA methyltransferase activities. Here we report that MBD1, a member of a conserved family of proteins able to bind methylated DNA, cooperates with PML-RARα in transcriptional repression and cellular transformation. PML-RARα recruits MBD1 to its target promoter through an HDAC3-mediated mechanism. Binding of HDAC3 and MBD1 is not confined to the promoter region but instead is spread over the locus. Knock-down of HDAC3 expression by RNA interference in acute promyelocytic leukemia cells alleviates PML-RAR-induced promoter silencing. We further demonstrate that retroviral expression of dominant-negative mutants of MBD1 in hematopoietic precursors compromises the ability of PML-RARα to block their differentiation and thus restored cell differentiation. Our results demonstrate that PML-RARα functions by recruiting an HDAC3-MBD1 complex that contributes to the establishment and maintenance of the silenced chromatin state.

MOZ-TIF2 Inhibits Transcription by Nuclear Receptors and p53 by Impairment of CBP Function

ABSTRACT Chromosomal rearrangements associated with acute myeloid leukemia (AML) include fusions of the genes encoding the acetyltransferase MOZ or MORF with genes encoding the nuclear receptor coactivator TIF2, p300, or CBP. Here we show that MOZ-TIF2 acts as a dominant inhibitor of the transcriptional activities of CBP-dependent activators such as nuclear receptors and p53. The dominant negative property of MOZ-TIF2 requires the CBP-binding domain (activation domain 1 [AD1]), and coimmunoprecipitation and fluorescent resonance energy transfer experiments show that MOZ-TIF2 interacts with CBP directly in vivo. The CBP-binding domain is also required for the ability of MOZ-TIF2 to extend the proliferative potential of murine bone marrow lineage-negative cells in vitro. We show that MOZ-TIF2 displays an aberrant nuclear distribution and that cells expressing this protein have reduced levels of cellular CBP, leading to depletion of CBP from PML bodies. In summary, our results indicate that disruption of the normal function of CBP and CBP-dependent activators is an important feature of MOZ-TIF2 action in AML.

In Vivo Genetic Screens of Patient-Derived Tumors Revealed Unexpected Frailty of the Transformed Phenotype

UNLABELLED The identification of genes maintaining cancer growth is critical to our understanding of tumorigenesis. We report the first in vivo genetic screen of patient-derived tumors, using metastatic melanomas and targeting 236 chromatin genes by expression of specific shRNA libraries. Our screens revealed unprecedented numerosity of genes indispensable for tumor growth (∼50% of tested genes) and unexpected functional heterogeneity among patients (<15% in common). Notably, these genes were not activated by somatic mutations in the same patients and are therefore distinguished from mutated cancer driver genes. We analyzed underlying molecular mechanisms of one of the identified genes, the Histone-lysine N-methyltransferase KMT2D, and showed that it promotes tumorigenesis by dysregulating a subset of transcriptional enhancers and target genes involved in cell migration. The assembly of enhancer genomic patterns by activated KMT2D was highly patient-specific, regardless of the identity of transcriptional targets, suggesting that KMT2D might be activated by distinct upstream signaling pathways. SIGNIFICANCE Drug targeting of biologically relevant cancer-associated mutations is considered a critical strategy to control cancer growth. Our functional in vivo genetic screens of patient-derived tumors showed unprecedented numerosity and interpatient heterogeneity of genes that are essential for tumor growth, but not mutated, suggesting that multiple, patient-specific signaling pathways are activated in tumors. Cancer Discov; 6(6); 650-63. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 561.

Functional characterization of a novel FGFR1OP-RET rearrangement in hematopoietic malignancies

The RET (REarranged during Transfection) receptor tyrosine kinase is targeted by oncogenic rearrangements in thyroid and lung adenocarcinoma. Recently, a RET (exon 12) rearrangement with FGFR1OP [fibroblast growth factor receptor 1 (FGFR1) oncogene partner] (exon 12) was identified in one chronic myelomonocytic leukemia (CMML) patient. We report the molecular cloning and functional characterization of a novel FGFR1OP (exon 11)‐RET (exon 11) gene fusion event (named FGFR1OP‐RET), mediated by a reciprocal translocation t(6; 10)(q27; q11), in a patient affected by primary myelofibrosis (PMF) with secondary acute myeloid leukemia (AML). The FGFR1OP‐RET fusion protein displayed constitutive tyrosine kinase and transforming activity in NIH3T3 fibroblasts, and induced IL3‐independent growth and activation of PI3K/STAT signaling in hematopoietic Ba/F3 cells. FGFR1OP‐RET supported cytokine‐independent growth, protection from stress and enhanced self‐renewal of primary murine hematopoietic progenitor and stem cells in vitro. In vivo, FGFR1OP‐RET caused a spectrum of disease phenotypes, with >50% of mice showing a fatal myeloproliferative disorder (MPD). Other phenotypes were leukemia transplantable in secondary recipients, dramatic expansion of the mast cell lineage, and reduction of repopulating activity upon lethal irradiation. In conclusion, FGFR1OP‐RET chimeric oncogenes are endowed with leukemogenic potential and associated to myeloid neoplasms (CMML and PMF/AML).

Dual modulation of MCL-1 and mTOR determines the response to sunitinib

Most patients who initially respond to treatment with the multi–tyrosine kinase inhibitor sunitinib eventually relapse. Therefore, developing a deeper understanding of the contribution of sunitinib’s numerous targets to the clinical response or to resistance is crucial. Here, we have shown that cancer cells respond to clinically relevant doses of sunitinib by enhancing the stability of the antiapoptotic protein MCL-1 and inducing mTORC1 signaling, thus evoking little cytotoxicity. Inhibition of MCL-1 or mTORC1 signaling sensitized cells to clinically relevant doses of sunitinib in vitro and was synergistic with sunitinib in impairing tumor growth in vivo, indicating that these responses are triggered as prosurvival mechanisms that enable cells to tolerate the cytotoxic effects of sunitinib. Furthermore, higher doses of sunitinib were cytotoxic, triggered a decline in MCL-1 levels, and inhibited mTORC1 signaling. Mechanistically, we determined that sunitinib modulates MCL-1 stability by affecting its proteasomal degradation. Dual modulation of MCL-1 stability at different dose ranges of sunitinib was due to differential effects on ERK and GSK3&bgr; activity, and the latter also accounted for dual modulation of mTORC1 activity. Finally, comparison of patient samples prior to and following sunitinib treatment suggested that increases in MCL-1 levels and mTORC1 activity correlate with resistance to sunitinib in patients.

RNAi screens identify CHD4 as an essential gene in breast cancer growth

Epigenetic regulation plays an essential role in tumor development and epigenetic modifiers are considered optimal potential druggable candidates. In order to identify new breast cancer vulnerabilities and improve therapeutic chances for patients, we performed in vivo and in vitro shRNA screens in a human breast cancer cell model (MCF10DCIS.com cell line) using epigenetic libraries. Among the genes identified in our screening, we deeply investigated the role of Chromodomain Helicase DNA binding Protein 4 (CHD4) in breast cancer tumorigenesis. CHD4 silencing significantly reduced tumor growth in vivo and proliferation in vitro of MCF10DCIS.com cells. Similarly, in vivo breast cancer growth was decreased in a spontaneous mouse model of breast carcinoma (MMTV-NeuT system) and in metastatic patient-derived xenograft models. Conversely, no reduction in proliferative ability of non-transformed mammary epithelial cells (MCF10A) was detected. Moreover, we showed that CHD4 depletion arrests proliferation by inducing a G0/G1 block of cell cycle associated with up-regulation of CDKN1A (p21). These results highlight the relevance of genetic screens in the identification of tumor frailties and the role of CHD4 as a potential pharmacological target to inhibit breast cancer growth.

WDR5 regulates epithelial-to-mesenchymal transition in breast cancer cells via TGFB

Even if the mortality rate in breast cancer (BC) has recently decreased, development of metastases and drug resistance are still challenges to successful systemic treatment. The epithelial-to-mesenchymal transition (EMT), as well as epigenetic dynamic modifications, plays a pivotal role in invasion, metastasis, and drug resistance. Here, we report that WDR5, the core subunit of histone H3 K4 methyltransferase complexes, is crucial in coordinating EMT and regulating epigenetic changes that drive metastasis. We show that silencing of WDR5 in BC up-regulates an epithelial signature in triple negative and luminal B like patients by transcriptional repression of mesenchymal genes and reduction of the metastatic properties of these cells. Moreover, we demonstrate that this regulation is mediated by inhibition of the TGFβ signaling both at the transcriptional and post-translational level, suggesting an active role of WDR5 in guiding tumor plasticity upon oncogenic insults, regardless of the pathological BC subtypes. We therefore suggest that WDR5 inhibition could be a successful pharmacologic approach to inhibit EMT and sensitize breast cancer cells to chemotherapy.

Abstract A28: Challenging new epigenetic vulnerabilities in human metastatic melanoma PDXs

Metastatic melanoma is one of the most aggressive cancers, refractory to most of the current therapies in its late stage. Melanoma is characterized by a strong molecular heterogeneity, and patients can be stratified in different subtypes according to genetic alterations. Despite the approval of new targeted drugs, patient treatment produces only partial responses and frequent and rapid relapses, mostly due to increased resistance. Mounting evidence highlights the necessity to better stratify melanomas on the basis of altered molecular pathways and to define new specific and effective tailored therapies. Our main goal is the genetic and functional characterization of metastatic melanomas aimed to generate new predictive and prognostic biomarkers and to identify new potential “druggable” candidates involved in melanoma progression. We have generated a series of human-in-mouse melanoma models, by xeno-transplantation of a cohort of human melanoma samples in immune-compromised mice. Our patient-derived xenografts (PDXs) faithfully recapitulate the heterogeneity of the original tumors at phenotypic and genetic level. Taking advantage of our model, we performed the first in vivo genetic screen on patient-derived tumors, using metastatic NRAS- and BRAF-mutant melanomas and targeting chromatin genes (~240). Our screens revealed unprecedented numerosity of tumor drivers (~50% of tested genes) and unexpected functional heterogeneity among patients (~60 drivers per tumor; Citation Format: Daniela Bossi, Angelo Cicalese, Ivan Gaetano Dellino, Lucilla Luzi, Simona Punzi, Carolina D9Alesio, Elena Cavallaro, Saverio Minucci, PierGiuseppe Pelicci, Luisa Lanfrancone. Challenging new epigenetic vulnerabilities in human metastatic melanoma PDXs. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr A28.

Abstract B43: PILOT: a patient-oriented in vivo functional platform to identify new lethalities and optimize cancer treatment

Large-scale genomics efforts have provided the opportunity to access a comprehensive catalog of genetic alterations in multiple cancers. However, it has also become apparent that very few driver mutations are emerging and as a consequence of that, limited opportunities exist to target mutated oncogenic proteins. It is imperative therefore to develop alternative approaches to therapy that can leverage the selective vulnerabilities of tumor cells resulting from the engagement of abnormal pathway connectivity. These can be best exploited in vivo, in a context that is closer to the environment tumors strive in. To identify new relevant actionable dependencies we have developed PILOT (Patient-oriented In vivo Lethality to Optimize Treatment), a loss-of-function in vivo platform for rapid identification of potential therapeutic targets in Patient-Derived Xenografts (PDXs). By optimizing primary tumor explant and expansion, determination of tumor-initiating cell frequency trough retroviral-mediated transduction, in vivo RNA interference screens, next-generation sequencing and analytic pipelines, we have been able to establish a comprehensive “patient-centric” approach oriented towards the identification of the highest priority genetic targets in specific clinico-pathological and mutational settings. So far, the main limitation for the systematic exploitation of in vivo functional genomics systems to elucidate patient vulnerabilities in the PDX models come from the limited number of human cells contributing to tumor establishment in a transplantation setting. The frequency of these tumors initiating cells (TICs) is commonly estimated by time-consuming limiting dilution assays and may consistently vary between different tumor origins. With this in mind, we have integrated in our platform a system based on scrambled barcoded libraries that allows to directly assess the required coverage of screening libraries in each model and adjust the RNAi screens for this factor. Our coverage study demonstrated to be a powerful tool to identify the minimal number of cells/barcode required to sustain a complex library in PDX models and at the same time a step forward to personalize the in vivo screening patient-by-patient. As proof of concept, we applied our PILOT platform to systematically interrogate context-specific epigenetic dependencies in pancreatic ductal adenocarcinoma (PDAC). In addition to the well-known genetic alterations (KRAS, TP53, CDKN2A/p16, SMAD4), some epigenetic mechanisms demonstrated to play a central role in PDAC progression and some of them could intriguingly represent new points of vulnerability, due to the low-frequency of mutation (ex. collateral or synthetic lethality). Our screening system utilized fully annotated low-passage PDAC xenografts and a lentiviral library of pooled shRNAs targeting 230 potentially “druggable” epigenetic regulators adjusted for the coverage study in each PDX. Hairpin-associated molecular barcodes were quantified by massively parallel sequencing and clustered according to their depletion or enrichment in comparison to a control population before transplantation. To date, we have completed a total of 5 in vivo screens using diverse PDAC xenograft models and, applying our comprehensive mutational and functional data analytics pipeline, we have developed a high-throughput validation scheme to triage “hits” that emerge from each screen. Focusing on epigenetic regulators, we identified WDR5, a core member of the COMPASS histone H3 Lys4 (H3K4) MLL (1-4) methyltransferase complex, as a top tumor maintenance hit required across multiple PDAC tumors and associated with the presence of G1-checkpoint alterations (p53 or p16). Mechanistically, WDR5 functions to sustain proper execution of DNA replication in PDAC cells, as previously suggested by replication stress studies involving MLL1, a critical ATR substrate, and c-Myc, also found to interact with WDR5. We indeed demonstrated that the WDR5-Myc interaction is critical for this replicative function and protects the PDAC cells from the excessive DNA damage accumulation and mitotic catastrophe. Intriguingly, this checkpoint function executed by the WDR5-Myc axis to protect the S-phase seems to be an addiction of the cancer cells, that have more active replication forks to stabilize in order to sustain the abnormal proliferative burst. To confirm this new cancer-associated lethality, we demonstrated that normal cells display less sensitivity to this replication checkpoint in virtue of their proficient G1-checkpoints and reduced time spent in S-phase compared to cancer cells. So, our PILOT platform was able to illuminate new therapeutic vulnerabilities that can be rapidly evaluated in the clinic through the development of WDR5-Myc inhibitors. In the near future, we plan to extend this platform in syngeneic mouse models, where one can probe the effects of target inhibition in the context of an intact immune response and in the presence of immune checkpoint activators, and in association with approved drugs, to identify new therapeutic options for recalcitrant tumor populations. Citation Format: Alessandro Carugo, Giannicola Genovese, Sahil Seth, Luigi Nezi, Angelo Cicalese, Daniela Bossi, Johnathon L. Rose, Andrea Viale, Luisa Lanfrancone, Timothy P. Heffernan, Giulio F. Draetta. PILOT: a patient-oriented in vivo functional platform to identify new lethalities and optimize cancer treatment. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr B43.