Daniel Massó-Vallés

Inhibition of Myc family proteins eradicates KRas-driven lung cancer in mice.

The principal reason for failure of targeted cancer therapies is the emergence of resistant clones that regenerate the tumor. Therapeutic efficacy therefore depends on not only how effectively a drug inhibits its target, but also the innate or adaptive functional redundancy of that target and its attendant pathway. In this regard, the Myc transcription factors are intriguing therapeutic targets because they serve the unique and irreplaceable role of coordinating expression of the many diverse genes that, together, are required for somatic cell proliferation. Furthermore, Myc expression is deregulated in most-perhaps all-cancers, underscoring its irreplaceable role in proliferation. We previously showed in a preclinical mouse model of non-small-cell lung cancer that systemic Myc inhibition using the dominant-negative Myc mutant Omomyc exerts a dramatic therapeutic impact, triggering rapid regression of tumors with only mild and fully reversible side effects. Using protracted episodic expression of Omomyc, we now demonstrate that metronomic Myc inhibition not only contains Ras-driven lung tumors indefinitely, but also leads to their progressive eradication. Hence, Myc does indeed serve a unique and nondegenerate role in lung tumor maintenance that cannot be complemented by any adaptive mechanism, even in the most aggressive p53-deficient tumors. These data endorse Myc as a compelling cancer drug target.

Myc inhibition is effective against glioma and reveals a role for Myc in proficient mitosis

Gliomas are the most common primary tumours affecting the adult central nervous system and respond poorly to standard therapy. Myc is causally implicated in most human tumours and the majority of glioblastomas have elevated Myc levels. Using the Myc dominant negative Omomyc, we previously showed that Myc inhibition is a promising strategy for cancer therapy. Here, we preclinically validate Myc inhibition as a therapeutic strategy in mouse and human glioma, using a mouse model of spontaneous multifocal invasive astrocytoma and its derived neuroprogenitors, human glioblastoma cell lines, and patient-derived tumours both in vitro and in orthotopic xenografts. Across all these experimental models we find that Myc inhibition reduces proliferation, increases apoptosis and remarkably, elicits the formation of multinucleated cells that then arrest or die by mitotic catastrophe, revealing a new role for Myc in the proficient division of glioma cells.

Ibrutinib Exerts Potent Antifibrotic and Antitumor Activities in Mouse Models of Pancreatic Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a dense stromal fibroinflammatory reaction that is a major obstacle to effective therapy. The desmoplastic stroma comprises many inflammatory cells, in particular mast cells as key components of the PDAC microenvironment, and such infiltration correlates with poor patient outcome. Indeed, it has been hypothesized that stromal ablation is critical to improve clinical response in patients with PDAC. Ibrutinib is a clinically approved Brutons tyrosine kinase inhibitor that inhibits mast cells and tumor progression in a mouse model of β-cell tumorigenesis. Here, we show that ibrutinib is highly effective at limiting the growth of PDAC in both transgenic mouse and patient-derived xenograft models of the disease. In these various experimental settings, ibrutinib effectively diminished fibrosis, extended survival, and improved the response to clinical standard-of-care therapy. Our results offer a preclinical rationale to immediately evaluate the clinical efficacy of ibrutinib in patients with PDAC.

Ibrutinib repurposing: from B-cell malignancies to solid tumors.

Ibrutinib (Imbruvica®, also known as PCI-32765) is a first-in-class, irreversible small-molecule inhibitor of Brutons Tyrosine Kinase (BTK) that binds covalently to cysteine C481 within the ATP-binding pocket. Since BTK is a Tec family non-receptor tyrosine kinase that is specifically required for B-cell antigen receptor (BCR) signaling, ibrutinib was initially developed for the treatment of B-cell malignancies. Currently, it is approved for first-line treatment of chronic lymphocytic leukemia, and for the treatment of mantle-cell lymphoma and Waldenstroms macroglobulinemia patients that have received at least one previous therapy. However, BTK is also involved in signaling pathways downstream of many other receptors and its expression is not restricted to B cells. In fact, it is expressed in all hematopoietic lineages with the exception of T lymphocytes. Taking advantage of this aspect of BTK biology, some groups, including ours, have exploited the utility of inhibiting BTK in different cell types other than B cells. In particular, we validated the critical role of BTK for mast cell degranulation in mouse models of pancreatic cancer, namely insulinoma [1] and pancreatic ductal adenocarcinoma (PDAC) [2]. Ibrutinib was effective in both scenarios, leading to vasculature collapse and tumor regression in insulinoma and to a potent and unexpected anti- fibrotic effect in PDAC, where it synergized with standard of care chemotherapy to extend mice survival. This latter observation suggests that the anti- fibrotic activity of ibrutinib could be beneficial also for the treatment of other fibrotic diseases, an aspect that deserves further studies. More recently, Gunderson and colleagues contributed to the understanding of ibrutinib therapeutic impact in PDAC by describing how BTK regulates B-cell and macrophage-mediated T-cell suppression and demonstrating that ibrutinib restores T-cell dependent anti-tumor responses and triggers growth inhibition [3]. These studies combined led to the initiation of a phase 2/3 clinical trial for the use of ibrutinib in combination with nab- paclitaxel and gemcitabine in metastatic PDAC. Importantly, emerging data obtained from various mouse models of cancer suggests that beneficial use of ibrutinib could extend beyond pancreatic cancer to other solid tumors. For example, myeloid-derived suppressor cells (MDSCs) express BTK and are present in the stroma of many different tumors, causing immunosuppression and evasion of anti-tumor immune responses. Consistently, treatment of breast cancer and melanoma mouse models with ibrutinib reduced the number of MDSCs in the spleen and the tumor, and combination therapy with anti-PD-L1 resulted in reduced mammary tumor growth [4]. Moreover, synergy of ibrutinib with immune checkpoint blockade has also been reported in mouse models of lymphoma, breast and colon cancer, but in a BTK-independent manner [5]. In this case, suppression of tumor growth was due to inhibition of the interleukin-2–inducible T-cell kinase (ITK), an enzyme required by Th2 T cells, allowing a shift of T-cell immune responses to a Th1 bias. However, off-target (BTK-independent) effects of ibrutinib are not limited to ITK, since its inhibitory activity affects several other kinases including Tec, EGFR, HER2, HER3, HER4, JAK3, Blk, Fgr, Hck, Lck, Yes/YES1, Bmx/Etk and Txk. While this lack of selectivity could explain some of its side effects, it could be exploited for the treatment of tumors without BTK dependency. Indeed, two recent reports exemplify this approach by showing promising in vitro data against EGFR-mutant or overexpressed non-small cell lung cancer (NSCLC) [6] and HER2-positive breast cancer. Finally, expression of BTK variants was found in cancer cells of breast, prostate [7] and colon tumors [8]. In all cases, its downregulation by RNAi or its inhibition by pharmacological means, including ibrutinib, leads to impaired proliferation and/or cell death. All these findings broaden the spectrum of tumor types potentially susceptible to treatment with ibrutinib. Its use against solid tumors, alone or in combination with standard therapies or novel immune-based approaches, is increasingly becoming a clinically viable option. Indeed, a number of clinical trials are already underway for the treatment of different solid tumor types, such as EGFR mutant NSCLC (http://ClinicalTrials.gov Identifier: NCT02321540), refractory stage IV cutaneous melanoma (NCT02581930), metastatic pancreatic adenocarcinoma (NCT02562898 and NCT02436668), localized prostate cancer (NCT02643667), advanced gastrointestinal and genitourinary tumors (NCT02599324), advanced carcinoid and pancreatic neuroendocrine tumors (NCT02575300) and other relapsed or refractory solid tumors including lung and breast (NCT02403271). Results of these trials will shed light on the applicability of ibrutinib in human patients beyond hematological malignancies and quickly open the way to new immediate clinical applications. Nevertheless, further work will be needed in order to establish, for every tumor type, the contribution of BTK-related responses versus inhibition of other kinases, either in tumor cells themselves or in the tumor stroma. Ibrutinib constitutes a prime example of therapeutic switching, but there are likely many other drugs already in clinical use that are candidates for repositioning, and could offer new, safe and easily-implementable opportunities for cancer patients.

BET inhibition is an effective approach against KRAS-driven PDAC and NSCLC

Effectively treating KRAS-driven tumors remains an unsolved challenge. The inhibition of downstream signaling effectors is a way of overcoming the issue of direct targeting of mutant KRAS, which has shown limited efficacy so far. Bromodomain and Extra-Terminal (BET) protein inhibition has displayed anti-tumor activity in a wide range of cancers, including KRAS-driven malignancies. Here, we preclinically evaluate the effect of BET inhibition making use of a new BET inhibitor, BAY 1238097, against Pancreatic Ductal Adenocarcinoma (PDAC) and Non-Small Cell Lung Cancer (NSCLC) models harboring RAS mutations both in vivo and in vitro. Our results demonstrate that BET inhibition displays significant therapeutic impact in genetic mouse models of KRAS-driven PDAC and NSCLC, reducing both tumor area and tumor grade. The same approach also causes a significant reduction in cell number of a panel of RAS-mutated human cancer cell lines (8 PDAC and 6 NSCLC). In this context, we demonstrate that while BET inhibition by BAY 1238097 decreases MYC expression in some cell lines, at least in PDAC cells its anti-tumorigenic effect is independent of MYC regulation. Together, these studies reinforce the use of BET inhibition and prompt the optimization of more efficient and less toxic BET inhibitors for the treatment of KRAS-driven malignancies, which are in urgent therapeutic need.

Abstract 2167: Preclinical validation of an Omomyc cell-penetrating peptide as a viable anti-Myc therapy

Deregulation of the MYC oncoprotein promotes tumorigenesis in most, if not all, cancers and is often associated with poor prognosis. However, targeting MYC has long been considered impossible based on the assumption that it would cause catastrophic side effects in normal tissues. Despite this general preconceived notion, we showed that MYC inhibition exerts extraordinary therapeutic impact in various genetic mouse models of cancer, and causes only mild, well-tolerated and reversible side effects. For these studies we employed the systemic and conditional expression of a dominant negative of MYC, called Omomyc, which we designed and validated, and that can inhibit MYC transactivation function both in vitro and in vivo. To date, Omomyc has only been considered a proof of principle, with any potential clinical application limited to gene therapy. Here we actually show that the 11 kDa Omomyc polypeptide spontaneously transduces into cancer cells, demonstrating unexpected cell-penetrating ability. Once inside the nuclei, the polypeptide effectively blocks MYC binding to its target DNA sites, interfering with MYC transcriptional regulation and halting cell proliferation. Moreover, intranasal (i.n.) administration of the Omomyc polypeptide in mice results in its rapid and persistent distribution to lungs, as well as to other organs (i.e. intestine, liver, kidneys and brain). Importantly, i.n. treatment of mice bearing either Non-Small-Cell-Lung-Cancer (NSCLC) or glioblastoma (GBM) with the Omomyc cell-penetrating peptide (OmomycCPP) significantly reduces tumor burden compared to their control counterparts. Notably, tumor regression is accompanied by significant reprogramming of the tumor microenvironment and tumor immune response. In summary, our data indicate that this novel generation of polypeptides represents a new opportunity to potentially inhibit MYC pharmacologically in a variety of malignant diseases. Citation Format: Marie-eve Beaulieu, Toni Jauset, Daniel Masso-Valles, Peter Rahl, Sandra Martinez-Martin, Loika Maltais, Mariano F. Zacarias-Fluck, Silvia Casacuberta, Erika Serrano del Pozo, Christopher Fiore, Laia Foradada, Matthew Guenther, Eduardo Romero Sanz, Marta Oteo Vives, Cynthia Tremblay, Martin Montagne, Miguel Angel Morcillo Alonso, Jonathan R. Whitfield, Pierre Lavigne, Laura Soucek. Preclinical validation of an Omomyc cell-penetrating peptide as a viable anti-Myc therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2167. doi:10.1158/1538-7445.AM2017-2167

Abstract B23: Pushing Myc inhibition towards the clinic by direct delivery of cell-penetrating peptides

Inhibiting Myc has long been regarded as a promising cancer treatment. However, clinical Myc inhibition was considered unfeasible due to its central role in normal proliferation and the difficulties of targeting a nuclear transcription factor. The expression of Omomyc (a Myc inhibitor derived from the dimerization and DNA-binding domain of Myc) in the KRasG12D non-small cell lung cancer (NSCLC) mouse model challenged these assumptions, as it resulted in dramatic tumor clearance with only limited and well tolerated side effects in normal tissues (Soucek et al., 2008 and 2013). Omomyc expression proved equally potent in several other mouse models of cancer, revealing the huge potential of this inhibitory approach against multiple cancer types including papilloma, pancreas and glioma (Soucek et al., 2004; Sodir et al., 2011; Annibali et al., 2014). Recently, Max*, a b-HLH-LZ peptide derived from Myc9s obligate protein partner Max, was shown to spontaneously enter cells (Montagne et al., 2012). As Omomyc and Max* display high structural homology, we hypothesized that Omomyc could also behave as a cell-penetrating peptide and thus recapitulate the effects of its transgenic counterpart. Our preliminary results show that the Omomyc peptide is well folded in solution; it transduces into cancer cells and effectively stops their proliferation in a dose-dependent manner.In vivo, nasal instillation of fluorescently-labeled Omomyc peptide leads to its rapid distribution to lungs and brain, as well as to other organs (G.I. tract, liver), as observed by IVIS® imaging and immunohistochemistry. Finally, a short treatment with the Omomyc peptide reduces the tumor size and number of Ki67 positive cells in the KRasG12D-induced NSCLC mouse model. In summary, the Omomyc cell penetrating peptide represents a new opportunity to pharmacologically inhibit Myc in a variety of malignant diseases. Citation Format: Marie-Eve Beaulieu, Toni Jauset, Daniel Masso-Valles, Jonathan R. Whitfield, Erika Serrano del Pozo, Cynthia Tremblay, Loika Maltais, Martin Montagne, Pierre Lavigne, Laura Soucek. Pushing Myc inhibition towards the clinic by direct delivery of cell-penetrating peptides. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr B23.

Abstract PR10: Preclinical validation of Myc inhibition by a new generation of Omomyc-based cell penetrating peptides

Deregulated Myc is associated with most human cancers suggesting that its inhibition would be a useful therapeutic strategy. Indeed, we have shown that Myc inhibition displays extraordinary therapeutic benefit in various transgenic mouse models of cancer (i.e skin, lung, pancreatic cancer and glioma) and causes only mild, well-tolerated and reversible side effects in normal tissues. For these studies we employed a dominant negative inhibitor of Myc, called Omomyc, which proved to be the most effective inhibitor of Myc transactivation function both in vitro and in vivo. Omomyc has so far been utilized exclusively as a transgene and served as a proof of principle. Here we report the exciting finding that Omomyc-based Cell Penetrating Peptides (CPPs) are a novel, state-of-the-art method for directly utilizing Omomyc itself (and similar peptides) to treat tumors in the lung and brain, where the peptides biodistribute after intranasal administration. We provide a comprehensive preclinical validation of this innovative therapeutic approach for pharmacological inhibition of Myc in cancer cell lines of different origin and genetic make-up, as well as in a mouse model of Non-Small-Cell Lung Cancer (NSCLC), where the Omomyc-CPPs, like their transgenic counterpart before, display a dramatic therapeutic impact. Citation Format: Marie-eve Beaulieu, Jonathan Whitfield, Daniel Masso-Valles, Toni Jauset, Erika Serrano, Martin Montagne, Loika Maltais, Cynthia Tremblay, Pierre Lavigne, Laura Soucek. Preclinical validation of Myc inhibition by a new generation of Omomyc-based cell penetrating peptides. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr PR10.

Abstract 2645: Preclinical validation of Myc inhibition by a new generation of Omomyc-based inhibitors

Deregulated Myc is associated with most human cancers suggesting that its inhibition would be a useful therapeutic strategy. Indeed, we have shown that Myc inhibition displays extraordinary therapeutic benefit in various transgenic mouse models of cancer (i.e. skin, lung, pancreatic cancer and glioma) and causes only mild, well-tolerated and reversible side effects in normal tissues. Furthermore, we demonstrated that Myc has a non-degenerate function in cancer that cannot be replaced by other pathways, even in the most aggressive p53-null tumors. Therefore, Myc could be targeted safely and successfully without eliciting resistance to therapy. For these studies we employed a dominant negative inhibitor of Myc, called Omomyc, which is an effective inhibitor of Myc transactivation function both in vitro and in vivo. Omomyc has so far been utilized exclusively as a transgene and served as a successful proof of principle. Here we discuss our current research with Omomyc and our efforts to develop a clinically viable approach to Myc inhibition. One is based on the direct use of Omomyc itself as a peptide since we have discovered that it natively possesses cell-penetrating activity and it rapidly biodistributes to the lung and brain after intranasal administration. We are finding that the Omomyc peptide - like its transgenic counterpart before - has a therapeutic impact and we are continuing with the preclinical validation of this innovative therapeutic approach to pharmacological Myc inhibition. The second approach takes advantage of state-of-the-art nanocarrier technology to deliver Omomyc systemically, that can be combined with tumour-targeting ligands. These two novel Myc inhibition strategies have the potential to be translated rapidly to the clinic. Citation Format: Marie-Eve Beaulieu, Toni Jauset, Daniel Masso-Valles, Jonathan Whitfield, Erika Serrano, Martin Montagne, Pierre Lavigne, Antonio Villaverde, Mireia Pesarrodona, Esther Vazquez, Laura Soucek. Preclinical validation of Myc inhibition by a new generation of Omomyc-based inhibitors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2645. doi:10.1158/1538-7445.AM2015-2645

Abstract 4956: Pharmacological inhibition of Bruton's Tyrosine Kinase (BTK) as a therapy for insulinoma and pancreatic ductal adenocarcinoma.

Myc, a pleiotropic transcription factor that is deregulated and/or over-expressed in most human cancers, instructs multiple extracellular programs that are required to sustain the complex microenvironment needed for tumor maintenance, including remodeling of tumor stroma, angiogenesis and inflammation. We previously showed in a model of pancreatic [[Unsupported Character - Symbol Font β]] cell tumorigenesis that acute Myc activation in vivo triggers rapid recruitment of mast cells to the tumor site and that this is absolutely required for angiogenesis and macroscopic tumor expansion. Moreover, systemic inhibition of mast cell degranulation with sodium cromoglycate induced death of tumor and endothelial cells in established tumors. Hence, mast cells are required both to establish and to maintain the tumors. While this intimates that selective inhibition of mast cell function could be therapeutically efficacious, cromoglycate is not a practical drug for systemic delivery in humans and no other systemic inhibitor of mast cell degranulation has hitherto been available. Ibrutinib is a novel inhibitor of Bruton9s tyrosine kinase (Btk) that blocks mast cell degranulation and is currently in clinical trials as a therapy for B cell non-Hodgkin9s lymphoma. Here, we show that systemic treatment of insulinoma-bearing mice with Ibrutinib efficiently inhibits Btk, blocks mast cell degranulation and triggers collapse of tumor vasculature and tumor regression. We also show that pancreatic ductal adenocarcinoma (PDAC)-bearing mice treated with Ibrutinib survive longer and present reduced tumor stroma, suggesting that combinatorial therapy with Ibrutinib and standard of care is a feasible therapeutic approach. Conclusions: our data reinforce the notion that mast cell function is required for maintenance of certain tumor types and indicate that the Btk inhibitor Ibrutinib may be useful in treating pancreatic cancer. Citation Format: Daniel Masso-Valles, Erika Serrano, Jonathan R. Whitfield, Joseph J. Buggy, Nicole M. Sodir, Nesrine I. Affara, Lamorna Brown Swigart, Gerard I. Evan, Laura Soucek. Pharmacological inhibition of Bruton9s Tyrosine Kinase (BTK) as a therapy for insulinoma and pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4956. doi:10.1158/1538-7445.AM2013-4956