Rene Figueredo

Use of the Humanized Anti-Epidermal Growth Factor Receptor Monoclonal Antibody h-R3 in Combination With Radiotherapy in the Treatment of Locally Advanced Head and Neck Cancer Patients

PURPOSE To evaluate safety and preliminary efficacy of the humanized anti-epidermal growth factor receptor monoclonal antibody h-R3 in combination with radiotherapy (RT) in unresectable head and neck cancer patients. Secondary end points were the measurement of h-R3 serum levels and the assessment of the potential mechanisms of antitumor effect on patient biopsies. Anti-idiotypic response to h-R3 was assessed. To predict pharmacologic effect, a mathematical model for antibodies recognizing antigens expressed in tumors and normal tissues was built. PATIENTS AND METHODS Twenty-four patients with advanced carcinomas of the head and neck received six once-weekly infusions of h-R3 at four dose levels in combination with RT. Pretreatment tumor biopsies were obtained to evaluate epidermal growth factor receptor expression as an enrollment criterion. Second biopsies were taken to evaluate the proliferative activity and angiogenesis in comparison with the pretreatment samples. Patient serum samples were collected to measure h-R3 levels and anti-idiotypic response. RESULTS The combination of h-R3 and RT was well tolerated. Antibody-related adverse events consisted in infusion reactions. No skin or allergic toxicity appeared. Overall survival significantly increased after the use of the higher antibody doses. Immunohistochemistry studies of tumor specimens before and after treatment revealed that antitumor response correlated with antiproliferative and antiangiogenic effect. One patient developed antibodies to h-R3. The mathematical model predicted that the maximum difference between the area under the curve in tumors and normal tissues is reached when the antibody has intermediate affinity. CONCLUSION h-R3 is a well-tolerated drug that may enhance radiocurability of unresectable head and neck neoplasms.

TTI-621 (SIRPαFc): A CD47-Blocking Innate Immune Checkpoint Inhibitor with Broad Anti-Tumor Activity and Minimal Erythrocyte Binding

Purpose: The ubiquitously expressed transmembrane glycoprotein CD47 delivers an anti-phagocytic (do not eat) signal by binding signal-regulatory protein α (SIRPα) on macrophages. CD47 is overexpressed in cancer cells and its expression is associated with poor clinical outcomes. TTI-621 (SIRPαFc) is a fully human recombinant fusion protein that blocks the CD47–SIRPα axis by binding to human CD47 and enhancing phagocytosis of malignant cells. Blockade of this inhibitory axis using TTI-621 has emerged as a promising therapeutic strategy to promote tumor cell eradication. Experimental Design: The ability of TTI-621 to promote macrophage-mediated phagocytosis of human tumor cells was assessed using both confocal microscopy and flow cytometry. In vivo antitumor efficacy was evaluated in xenograft and syngeneic models and the role of the Fc region in antitumor activity was evaluated using SIRPαFc constructs with different Fc tails. Results: TTI-621 enhanced macrophage-mediated phagocytosis of both hematologic and solid tumor cells, while sparing normal cells. In vivo, TTI-621 effectively controlled the growth of aggressive AML and B lymphoma xenografts and was efficacious in a syngeneic B lymphoma model. The IgG1 Fc tail of TTI-621 plays a critical role in its antitumor activity, presumably by engaging activating Fcγ receptors on macrophages. Finally, TTI-621 exhibits minimal binding to human erythrocytes, thereby differentiating it from CD47 blocking antibodies. Conclusions: These data indicate that TTI-621 is active across a broad range of human tumors. These results further establish CD47 as a critical regulator of innate immune surveillance and form the basis for clinical development of TTI-621 in multiple oncology indications. Clin Cancer Res; 23(4); 1068–79. ©2016 AACR.

BRCA2 inhibition enhances cisplatin-mediated alterations in tumor cell proliferation, metabolism, and metastasis

Tumor cells have unstable genomes relative to non‐tumor cells. Decreased DNA integrity resulting from tumor cell instability is important in generating favorable therapeutic indices, and intact DNA repair mediates resistance to therapy. Targeting DNA repair to promote the action of anti‐cancer agents is therefore an attractive therapeutic strategy. BRCA2 is involved in homologous recombination repair. BRCA2 defects increase cancer risk but, paradoxically, cancer patients with BRCA2 mutations have better survival rates. We queried TCGA data and found that BRCA2 alterations led to increased survival in patients with ovarian and endometrial cancer. We developed a BRCA2‐targeting second‐generation antisense oligonucleotide (ASO), which sensitized human lung, ovarian, and breast cancer cells to cisplatin by as much as 60%. BRCA2 ASO treatment overcame acquired cisplatin resistance in head and neck cancer cells, but induced minimal cisplatin sensitivity in non‐tumor cells. BRCA2 ASO plus cisplatin reduced respiration as an early event preceding cell death, concurrent with increased glucose uptake without a difference in glycolysis. BRCA2 ASO and cisplatin decreased metastatic frequency in vivo by 77%. These results implicate BRCA2 as a regulator of metastatic frequency and cellular metabolic response following cisplatin treatment. BRCA2 ASO, in combination with cisplatin, is a potential therapeutic anti‐cancer agent.

Combining Small Interfering RNAs Targeting Thymidylate Synthase and Thymidine Kinase 1 or 2 Sensitizes Human Tumor Cells to 5-Fluorodeoxyuridine and Pemetrexed

Thymidylate synthase (TS) is the only de novo source of thymidylate (dTMP) for DNA synthesis and repair. Drugs targeting TS protein are a mainstay in cancer treatment, but off-target effects and toxicity limit their use. Cytosolic thymidine kinase (TK1) and mitochondrial thymidine kinase (TK2) contribute to an alternative dTMP-producing pathway, by salvaging thymidine from the tumor milieu, and may modulate resistance to TS-targeting drugs. Combined down-regulation of these enzymes is an attractive strategy to enhance cancer therapy. We have shown previously that antisense-targeting TS enhanced tumor cell sensitivity to TS-targeting drugs in vitro and in vivo. Because both TS and TKs contribute to increased cellular dTMP, we hypothesized that TKs mediate resistance to the capacity of TS small interfering RNA (siRNA) to sensitize tumor cells to TS-targeting anticancer drugs. We assessed the effects of targeting TK1 or TK2 with siRNA alone and in combination with siRNA targeting TS and/or TS-protein targeting drugs on tumor cell proliferation. Down-regulation of TK with siRNA enhanced the capacity of TS siRNA to sensitize tumor cells to traditional TS protein-targeting drugs [5-fluorodeoxyuridine (5FUdR) and pemetrexed]. The sensitization was greater than that observed in response to any siRNA used alone and was specific to drugs targeting TS. Up-regulation of TK1 in response to combined 5FUdR and TS siRNA suggests that TK knockdown may be therapeutically useful in combination with these agents. TKs may be useful targets for cancer therapy when combined with molecules targeting TS mRNA and TS protein.

ODN 491, a novel antisense oligodeoxynucleotide that targets thymidylate synthase, exerts cell-specific effects in human tumor cell lines.

Thymidylate synthase (TS) is essential for DNA replication and is a target for cancer chemotherapy. However, toxicity to normal cells and tumor cell drug resistance necessitate development of new therapeutic strategies. One such strategy is to use antisense (AS) technology to reduce TS mRNA and protein levels in treated cells. We have developed oligodeoxynucleotides (ODNs) that target different regions of TS mRNA, inhibit human tumor cell proliferation as single agents, and enhance cytotoxicity of clinically useful TS protein-targeting drugs. Here we describe ODN 491, a novel 20mer AS ODN complementary to a previously untargeted portion of the TS mRNA coding region. AS ODN 491 decreased TS mRNA levels to different degrees in a panel of human tumor-derived cell lines, and induced different physiological effects in a tumor cell line-dependent manner. ODN 491 (like AS TS ODN 83, previously shown to be effective) decreased TS protein levels in HeLa cells with a concomitant increase in sensitivity to TS-targeting chemotherapeutics. However (and contrary to HeLa cell response to an AS ODN 83), it did not, as a single agent, inhibit HeLa cell proliferation. In MCF-7 cells, ODN 491 treatment was less effective at reducing TS mRNA and did not reduce TS protein, nor did it enhance sensitivity to TS-targeting or other chemotherapeutics. Moreover, specifically in MCF-7 cells but not HeLa cells, ODN 491 as a single agent induced apoptosis. These data indicate that AS TS ODN 491 is an effective AS reagent targeting a novel TS mRNA region. However, treatment of tumor cell lines with AS TS ODNs targeting different TS mRNA regions results in a pattern of physiological effects that varies in a tumor cell line-specific fashion. In addition, the capacity of different AS TS ODNs to induce physiological effects does not correlate well with their capacity to reduce TS mRNA and/or protein and, further, depends on the region of TS mRNA selected for targeting. Recognition of tumor cell-specific and mRNA region-specific variability in response to AS TS ODNs will be important in designing AS TS ODNs for potential clinical use.

Reciprocal positive selection for weakness - preventing olaparib resistance by inhibiting BRCA2

Human tumor heterogeneity promotes therapeutic failure by increasing the likelihood of resistant cell subpopulations. The PARP-1 inhibitor olaparib is approved for use in BRCA-mutated ovarian cancers but BRCA2-reversion mutations lead to functional homologous recombination repair (HRR) and olaparib resistance. To overcome that resistance and expand use of PARP1 inhibition to cancers with functional HRR, we developed an antisense strategy to render the majority of tumor cells in a population BRCA2-deficient. We predicted that this strategy would render HRR-proficient tumor cells sensitive to olaparib and prevent emergence of resistance in a tumor cell population heterogeneous for HRR proficiency. We report that BRCA2 downregulation sensitized multiple human tumor cell lines (but not non-cancer human kidney cells) to olaparib and, combined with olaparib, increased aneuploidy and chromosomal translocations in human tumor cells. In a mixed HRR-proficient and HRR-deficient cell population, olaparib monotherapy allowed outgrowth of HRR-proficient cells resistant to subsequent olaparib treatment. Combined BRCA2 inhibition and olaparib treatment prevented selection of HRR-proficient cells and inhibited proliferation of the entire population. Treatment with BRCA2 siRNA and olaparib decreased ovarian xenograft growth in mice more effectively than either treatment alone. In vivo use of BRCA2 antisense oligonucleotides may be a viable option to expand clinical use of olaparib and prevent resistance.

Synergistic cytotoxicity against human tumor cell lines by oncolytic adenovirus dl1520 (ONYX-015) and melphalan.

Aims and background In light of the need for more selective anticancer therapy, much work has been directed at developing compounds or biological agents that target functions specific to cancer cells. To this end, numerous viruses have been engineered to exploit the dependence of cancer cells on particular anomalies that contribute to their rogue proliferative activity, such as dysfunctional p53, overactive mitogenic signaling, or a defective interferon response. The oncolytic human adenovirus dl1520 (ONYX-015) was engineered to propagate specifically in p53-deficient tumors, which comprise over half of all tumors. Based on successes in clinical trials, the full potential of dl1520 and other oncolytic viruses may be even better realized by using them in combination with conventional chemotherapy drugs. Methods As a model system in which to test this potential, representative cell lines from 2 common cancer types, oral squamous cell carcinoma (HN-5a) and colon adenocarcinoma (HT-29), were chosen, as well as platinum-drug-resistant variants of each. Results Following preliminary screening of virus and drug combinations, dl1520 and melphalan were found to synergistically inhibit proliferation of all the cancer cell lines. Melphalan pretreatment or cotreatment with dl1520 enhanced inhibition of proliferation by dl1520 by up to 60% and increased apoptosis by up to 25%. The tight-junction protein CAR (coxsackie and adenovirus receptor), via which adenovirus enters cells, was not upregulated by treatment with melphalan, suggesting that other mechanisms contribute to synergy. Conclusions The synergy between melphalan and dl1520 suggests that tumor-selective cell killing by oncolytic viruses may be augmented by combining with cytotoxic drugs.

Abstract 3765: Indoleamine 2,3-dioxygenase mediates immune-independent human tumor cell resistance to olaparib, γ radiation, and cisplatin

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Indoleamine 2,3-dioxygenase-1 (IDO) is an immunosuppressive molecule expressed by most human tumors. IDO levels correlate with poor prognosis in cancer patients and IDO inhibitors are under investigation to enhance endogenous anticancer immunosurveillance. Little is known of immune-independent functions of IDO relevant to cancer therapy. We show, for the first time, that IDO mediates human tumor cell resistance to a PARP inhibitor (olaparib), γ radiation, cisplatin, and combined treatment with olaparib and radiation, in the absence of immune cells. Antisense-mediated reduction of IDO, alone and (in a synthetic lethal approach) in combination with antisense to the DNA repair protein BRCA2 sensitizes human lung cancer cells to olaparib and cisplatin. Antisense reduction of IDO decreased NAD+ in human tumor cells. NAD+ is essential for PARP activity and these data suggest that IDO mediates treatment resistance independent of immunity and at least partially due to a previously unrecognized role for IDO in DNA repair. Furthermore, IDO levels correlated with accumulation of tumor cells in G1 and depletion of cells in G2/M of the cell cycle, suggesting that IDO effects on cell cycle may also modulate sensitivity to radiation and chemotherapeutic agents. IDO is a potentially valuable therapeutic target in cancer treatment, independent of immune function and in combination with other therapies. Supported by grants to JK and WM from the Canadian Institutes of Health Research (CIHR). Citation Format: Saman Maleki Vareki, Mateusz Rytelewski, Rene Figueredo, Di Chen, Peter J. Ferguson, Mark Vincent, Weiping Min, Xiufen Zheng, James Koropatnick. Indoleamine 2,3-dioxygenase mediates immune-independent human tumor cell resistance to olaparib, γ radiation, and cisplatin. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3765. doi:10.1158/1538-7445.AM2014-3765

Abstract C77: A novel BRCA2 targeting antisense oligonucleotide sensitizes human tumor cells to chemotherapy and radiotherapy - the induction of ‘complementary lethality’ by targeting DNA repair.

BRCA2 is a protein involved in homologous recombination repair of double-stranded DNA breaks in human cells. Inactivating mutations in BRCA2 predispose to early onset cancer of the breast, ovary and other tissues. However, patients with tumors that harbour BRCA2 mutations respond more favourably to cancer therapy. Therefore, inhibiting BRCA2 function in cancer cells capable of homologous recombination repair may sensitize otherwise resistant tumors to distinct types of anti-cancer treatment. We have reported that BRCA2 siRNA sensitizes human tumor cells to specific DNA-damaging agents (e.g., cisplatin), but not anti-folate drugs (e.g., pemetrexed), a phenomenon we termed ‘complementary lethality’. Based on these results, we developed and are testing a second generation antisense oligodeoxynucleotide (BR-1) that specifically targets BRCA2. BR-1 decreased BRCA2 mRNA and protein levels, and also inhibited BRCA2-mediated RAD51 repair focus formation. BR-1 potently sensitized human non-small cell lung cancer (A549) cells to cisplatin, melphalan, and carboplatin as evidenced by reduced overall proliferation. In addition, A549 cells exhibited reduced ability to form colonies after concomitant treatment with BR-1 and cisplatin, melphalan, or ionizing radiation. Furthermore, cisplatin-resistant, patient-derived head and neck squamous cancer cells (HN-5a) were rendered sensitive to cisplatin following BR-1 treatment, and the level of cisplatin sensitivity was comparable to that of the parent population. Interestingly, treatment with BR-1 and cisplatin significantly decreased cellular respiration compared to control oligonucleotide and cisplatin treatment, suggesting that downregulation of BRCA2 in the context of alkylating drug treatment alters cellular metabolism. However, this change in respiration occurred independently of mitochondrial integrity. BR-1 is currently being tested in vivo to determine its ability to downregulate BRCA2 in human xenografts and sensitize solid tumors to chemotherapy. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C77. Citation Format: Mateusz Rytelewski, Jessica Tong, Adrian Buensucesco, Saman Maleki Vareki, Peter J. Ferguson, Rene Figueredo, Mark Vincent, Trevor Shepherd, Bonnie J. Deroo, James D. Koropatnick. A novel BRCA2 targeting antisense oligonucleotide sensitizes human tumor cells to chemotherapy and radiotherapy - the induction of ‘complementary lethality’ by targeting DNA repair. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C77.

Abstract C143: Combining siRNAs targeting thymidylate synthase and thymidine kinase 1 or 2 sensitizes human tumor cells to 5FUdR and pemetrexed.

Thymidylate synthase (TS) is the only de novo source of thymidylate (dTMP) for DNA synthesis and repair. Drugs targeting TS protein are a mainstay in cancer treatment, but off-target effects and toxicity limit their use. Cytosolic thymidine kinase (TK1) and mitochondrial thymidine kinase (TK2) contribute to an alternative dTMP-producing pathway, by salvaging thymidine from the tumor milieu, and may modulate resistance to TS-targeting drugs. Combined downregulation of these enzymes is an attractive strategy to enhance cancer therapy. We have previously shown that antisense targeting TS enhanced tumor cell sensitivity to TS-targeting drugs in vitro and in vivo. As both TS and TKs contribute to increased cellular dTMP, we hypothesized that TKs mediate resistance to the capacity of TS siRNA to sensitize tumor cells to TS-targeting anticancer drugs. We assessed the effects of targeting TK1 or TK2 with siRNA alone and in combination with siRNA targeting TS and/or TS-protein targeting drugs on tumor cell proliferation. Downregulation of TK with siRNA enhanced the capacity of TS siRNA to sensitize tumor cells to traditional TS protein-targeting drugs (5FUdR and pemetrexed). The sensitization was greater than that observed in response to any siRNA used alone, and was specific to drugs targeting TS. Upregulation of TK1 in response to combined 5FUdR and TS siRNA suggests that TK knockdown may be therapeutically useful in combination with these agents. TKs may be useful targets for cancer therapy when combined with molecules targeting TS mRNA and TS protein. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C143.