Derek Clements

Gemcitabine enhances the efficacy of reovirus-based oncotherapy through anti-tumour immunological mechanisms

Background:Reovirus preferentially infects and kills cancer cells and is currently undergoing clinical trials internationally. While oncolysis is the primary mode of tumour elimination, increasing evidence illustrates that reovirus additionally stimulates anti-tumour immunity with a capacity to target existing and possibly relapsing cancer cells. These virus-induced anti-tumour immune activities largely determine the efficacy of oncotherapy. On the other hand, anti-viral immune responses can negatively affect oncotherapy. Hence, the strategic management of anti-tumour and anti-viral immune responses through complementary therapeutics is crucial to achieve the maximum anti-cancer benefits of oncotherapy.Methods:Intra-peritoneal injection of mouse ovarian surface epithelial cells (ID8 cells) into wild-type C57BL/6 mice was treated with a therapeutic regimen of reovirus and/or gemcitabine and then analysed for prolonged survival, disease pathology, and various immunological parameters. Furthermore, in vitro analyses were conducted to assess apoptosis, viral spread, and viral production during reovirus and/or gemcitabine treatment.Results:We demonstrate that reovirus and gemcitabine combination treatment postpones peritoneal carcinomatosis development and prolongs the survival of cancer-bearing hosts. Importantly, these anti-cancer benefits are generated through various immunological mechanisms, including: (1) inhibition of myeloid-derived suppressor cells recruitment to the tumour microenvironment, (2) downmodulation of pro-MDSC factors, and (3) accelerated development of anti-tumour T-cell responses.Conclusion:The complementation of reovirus with gemcitabine further potentiates virus-initiated anti-cancer immunity and enhances the efficacy of oncotherapy. In the context of ongoing clinical trials, our findings represent clinically relevant information capable of enhancing cancer outcomes.

Multifaceted Therapeutic Targeting of Ovarian Peritoneal Carcinomatosis Through Virus-induced Immunomodulation

Immunosuppression associated with ovarian cancer (OC) and resultant peritoneal carcinomatosis (PC) hampers the efficacy of many promising treatment options, including immunotherapies. It is hypothesized that oncolytic virus-based therapies can simultaneously kill OC and mitigate immunosuppression. Currently, reovirus-based anticancer therapy is undergoing phase I/II clinical trials for the treatment of OC. Hence, this study was focused on characterizing the effects of reovirus therapy on OC and associated immune microenvironment. Our data shows that reovirus efficiently killed OC cells and induced higher expression of the molecules involved in antigen presentation including major histocompatibility complex (MHC) class I, β2-microglobulin (β2M), TAP-1, and TAP-2. In addition, in the presence of reovirus, dendritic cells (DCs) overcame the OC-mediated phenotypic suppression and successfully stimulated tumor-specific CD8+ T cells. In animal studies, reovirus targeted local and distal OC, alleviated the severity of PC and significantly prolonged survival. These therapeutic effects were accompanied by decreased frequency of suppressive cells, e.g., Gr1.1+, CD11b+ myeloid derived suppressor cells (MDSCs), and CD4+, CD25+, FOXP3+ Tregs, tumor-infiltration of CD3+ cells and higher expression of Th1 cytokines. Finally, reovirus therapy during early stages of OC also resulted in the postponement of PC development. This report elucidates timely information on a therapeutic approach that can target OC through clinically desired multifaceted mechanisms to better the outcomes.

Core needle biopsy of breast cancer tumors increases distant metastases in a mouse model.

INTRODUCTION: Incisional biopsies, including the diagnostic core needle biopsy (CNB), routinely performed before surgical excision of breast cancer tumors are hypothesized to increase the risk of metastatic disease. In this study, we experimentally determined whether CNB of breast cancer tumors results in increased distant metastases and examine important resultant changes in the primary tumor and tumor microenvironment associated with this outcome. METHOD: To evaluate the effect of CNB on metastasis development, we implanted murine mammary 4T1 tumor cells in BALB/c mice and performed CNB on palpable tumors in half the mice. Subsequently, emulating the human scenario, all mice underwent complete tumor excision and were allowed to recover, with attendant metastasis development. Tumor growth, lung metastasis, circulating tumor cell (CTC) levels, variation in gene expression, composition of the tumor microenvironment, and changes in immunologic markers were compared in biopsied and non-biopsied mice. RESULTS: Mice with biopsied tumors developed significantly more lung metastases compared to non-biopsied mice. Tumors from biopsied mice contained a higher frequency of myeloid-derived suppressor cells (MDSCs) accompanied by reduced CD4 + T cells, CD8 + T cells, and macrophages, suggesting biopsy-mediated development of an increasingly immunosuppressive tumor microenvironment. We also observed a CNB-dependent up-regulation in the expression of SOX4, Ezh2, and other key epithelial-mesenchymal transition (EMT) genes, as well as increased CTC levels among the biopsy group. CONCLUSION: CNB creates an immunosuppressive tumor microenvironment, increases EMT, and facilitates release of CTCs, all of which likely contribute to the observed increase in development of distant metastases.

Gemcitabine-mediated tumour regression and p53-dependent gene expression: implications for colon and pancreatic cancer therapy.

Gemcitabine is a chemotherapeutic that is widely used for the treatment of a variety of haematological malignancies and has become the standard chemotherapy for the treatment of advanced pancreatic cancer. Combinational gemcitabine regimes (e.g.with doxorubicin) are being tested in clinical trials to treat a variety of cancers, including colon cancer. The limited success of these trials has prompted us to pursue a better understanding of gemcitabine’s mechanism of cell killing, which could dramatically improve the therapeutic potential of this agent. For comparison, we included gamma irradiation that triggers robust cell cycle arrest and Cr(VI), which is a highly toxic chemical that induces a robust p53-dependent apoptotic response. Gemcitabine induced a potent p53-dependent apoptosis that correlated with the accumulation of pro-apoptotic proteins such as PUMA and Bax. This is accompanied by a drastic reduction in p2l and 14-3-3σ protein levels, thereby significantly sensitizing the cells to apoptosis. In vitro and in vivo studies demonstrated that gemcitabine required PUMA transcription to instigate an apoptotic programme. This was in contrast to Cr(VI)-induced apoptosis that required Bax and was independent of transcription. An examination of clinical colon and pancreatic cancer tissues shows higher p53, p21, 14-3-3σ and Bax expression compared with matched normal tissues, yet there is a near absence of PUMA protein. This may explain why gemcitabine shows only limited efficacy in the treatment of these cancers. Our results raise the possibility that targeting the Bax-dependent cell death pathway, rather than the PUMA pathway, could result in significantly improved patient outcome and prognosis for these cancers.

The NAD + salvage pathway modulates cancer cell viability via p73

The involvement of the nicotinamide adenine dinucleotide (NAD+) salvage pathway in cancer cell survival is poorly understood. Here we show that the NAD+ salvage pathway modulates cancer cell survival through the rarely mutated tumour suppressor p73. Our data show that pharmacological inhibition or knockdown of nicotinamide phosphoribosyltransferase (NAMPT), a rate-limiting enzyme in the NAD+ salvage pathway, enhances autophagy and decreases survival of cancer cells in a p53-independent manner. Such NAMPT inhibition stabilizes p73 independently of p53 through increased acetylation and decreased ubiquitination, resulting in enhanced autophagy and cell death. These effects of NAMPT inhibition can be effectively reversed using nicotinamide mononucleotide (NMN), the enzymatic product of NAMPT. Similarly, knockdown of p73 also decreases NAMPT inhibition-induced autophagy and cell death, whereas overexpression of p73 alone enhances these effects. We show that the breast cancer cell lines (MCF-7, MDA-MB-231 and MDA-MB-468) harbour significantly higher levels of NAMPT and lower levels of p73 than does the normal cell line (MCF-10A), and that NAMPT inhibition is cytotoxic exclusively to the cancer cells. Furthermore, data from 176 breast cancer patients demonstrate that higher levels of NAMPT and lower levels of p73 correlate with poorer patient survival, and that high-grade tumours have significantly higher NAMPT/p73 mRNA ratios. Therefore, the inverse relationship between NAMPT and p73 demonstrable in vitro is also reflected from the clinical data. Taken together, our studies reveal a new NAMPT-p73 nexus that likely has important implications for cancer diagnosis, prognosis and treatment.

Two is better than one: Complementing oncolytic virotherapy with gemcitabine to potentiate antitumor immune responses.

Oncolytic viruses (OVs) preferentially infect and kill cancer cells. Additionally, OV-induced immune responses subvert cancer-associated immunosuppression and promote antitumor immunity. We have recently demonstrated that the complementation of oncolytic virotherapy with gemcitabine accentuates its immunostimulatory effects, hence exerting superior antineoplastic activity.

Newly Recruited CD11b+, GR-1+, Ly6Chigh Myeloid Cells Augment Tumor-Associated Immunosuppression Immediately following the Therapeutic Administration of Oncolytic Reovirus

Tumor-associated immunosuppression aids cancer cells to escape immune-mediated attack and subsequent elimination. Recently, however, many oncolytic viruses, including reovirus, have been reported to overturn such immunosuppression and promote the development of a clinically desired antitumor immunity, which is known to promote favorable patient outcomes. Contrary to this existing paradigm, in this article we demonstrate that reovirus augments tumor-associated immunosuppression immediately following its therapeutic administration. Our data show that reovirus induces preferential differentiation of highly suppressive CD11b+, Gr-1+, Ly6Chigh myeloid cells from bone marrow hematopoietic progenitor cells. Furthermore, reovirus administration in tumor-bearing hosts drives time-dependent recruitment of CD11b+, Gr-1+, Ly6Chigh myeloid cells in the tumor milieu, which is further supported by virus-induced increased expression of numerous immune factors involved in myeloid-derived suppressor cell survival and trafficking. Most importantly, CD11b+, Gr-1+, Ly6Chigh myeloid cells specifically potentiate the suppression of T cell proliferation and are associated with the absence of IFN-γ response in the tumor microenvironment early during oncotherapy. Considering that the qualitative traits of a specific antitumor immunity are largely dictated by the immunological events that precede its development, our findings are of critical importance and must be considered while devising complementary interventions aimed at promoting the optimum efficacy of oncolytic virus–based anticancer immunotherapies.

Reovirus in cancer therapy: an evidence-based review

Reovirus, a double-stranded ribonucleic acid virus and benign human pathogen, preferentially infects and kills cancer cells in its unmodified form, and is one of the leading oncolytic viruses currently undergoing clinical trials internationally. With 32 clinical trials completed or ongoing thus far, reovirus has demonstrated clinical therapeutic applicability against a multitude of cancers, including but not limited to breast cancer, prostate cancer, pancreatic cancer, malignant gliomas, advanced head and neck cancers, and metastatic ovarian cancers. Phase I trials have demonstrated that reovirus is safe to use via both intralesional/intratumoral and systemic routes of administration, with the most common adverse reactions being grade I/II toxicities, such as flu-like illness (fatigue, nausea, vomiting, headache, fever/chills, dizziness), diarrhea, and lymphopenia. In subsequent Phase II trials, reovirus administration was demonstrated to successfully decrease tumor size and promote tumor necrosis, thereby complementing compelling preclinical evidence of tumor destruction by the virus. Importantly, reovirus has been shown to be effective as a monotherapy, as well as in combination with other anticancer options, including radiation and chemotherapeutic agents, such as gemcitabine, docetaxel, paclitaxel, and carboplatin. Of note, the first Phase III clinical trial using reovirus in combination with paclitaxel and carboplatin for the treatment of head and neck cancers is under way. Based on the evidence from clinical trials, we comprehensively review the use of reovirus as an anticancer agent, acknowledge key obstacles, and suggest future directions to ultimately potentiate the efficacy of reovirus oncotherapy.

Dendritic Cells in Oncolytic Virus-Based Anti-Cancer Therapy

Dendritic cells (DCs) are specialized antigen-presenting cells that have a notable role in the initiation and regulation of innate and adaptive immune responses. In the context of cancer, appropriately activated DCs can induce anti-tumor immunity by activating innate immune cells and tumor-specific lymphocytes that target cancer cells. However, the tumor microenvironment (TME) imposes different mechanisms that facilitate the impairment of DC functions, such as inefficient antigen presentation or polarization into immunosuppressive DCs. These tumor-associated DCs thus fail to initiate tumor-specific immunity, and indirectly support tumor progression. Hence, there is increasing interest in identifying interventions that can overturn DC impairment within the TME. Many reports thus far have studied oncolytic viruses (OVs), viruses that preferentially target and kill cancer cells, for their capacity to enhance DC-mediated anti-tumor effects. Herein, we describe the general characteristics of DCs, focusing on their role in innate and adaptive immunity in the context of the TME. We also examine how DC-OV interaction affects DC recruitment, OV delivery, and anti-tumor immunity activation. Understanding these roles of DCs in the TME and OV infection is critical in devising strategies to further harness the anti-tumor effects of both DCs and OVs, ultimately enhancing the efficacy of OV-based oncotherapy.

The NAD+ synthesizing enzyme nicotinamide mononucleotide adenylyltransferase 2 (NMNAT-2) is a p53 downstream target.

NAD+ metabolism plays key roles not only in energy production but also in diverse cellular physiology. Aberrant NAD+ metabolism is considered a hallmark of cancer. Recently, the tumor suppressor p53, a major player in cancer signaling pathways, has been implicated as an important regulator of cellular metabolism. This notion led us to examine whether p53 can regulate NAD+ biosynthesis in the cell. Our search resulted in the identification of nicotinamide mononucleotide adenylyltransferase 2 (NMNAT-2), a NAD+ synthetase, as a novel downstream target gene of p53. We show that NMNAT-2 expression is induced upon DNA damage in a p53-dependent manner. Two putative p53 binding sites were identified within the human NMNAT-2 gene, and both were found to be functional in a p53-dependent manner. Furthermore, knockdown of NMNAT-2 significantly reduces cellular NAD+ levels and protects cells from p53-dependent cell death upon DNA damage, suggesting an important functional role of NMNAT-2 in p53-mediated signaling. Our demonstration that p53 modulates cellular NAD+ synthesis is congruent with p53’s emerging role as a key regulator of metabolism and related cell fate.