Overcoming Innate and Acquired Therapy Resistance by Targeting DNA Repair in Human Cancer Cells

Genomic instability and a high mutation rate lead to heterogeneity in human tumors. Mathematical modelling predicts that these characteristics promote acquired resistance to cytotoxic and targeted therapies, by increasing the likelihood that resistant subpopulations exist at the start of treatment (and promoting the accumulation of de novo resistance mutations during treatment). As a result, genome plasticity promotes increased fitness on the population level, but individual tumor cells must nonetheless maintain a level of DNA integrity that allows for continued survival, particularly in the context of DNA-damaging therapy (which DNA repair counteracts). Thus, DNA repair proteins are a source of innate resistance to many common anti-cancer drugs, and represent intriguing targets for therapeutic attack. One way to forestall treatment resistance is to sensitize tumor cells to DNA-damaging therapy by inhibiting DNA repair and decreasing survival following drug treatment. I developed the concept of complementary lethality’, defined as “potentiation of drug therapy by the inhibition of DNA repair factors responsible for resistance to that specific drug”. BRCA2 is involved in homologous recombination repair (HRR) of double stranded breaks (DSBs) in DNA, and mutations in the BRCA2 gene predispose to various cancers. However, patients with BRCA2-mutated tumors respond more favourably to therapy. I found that inhibition of BRCA2 with siRNA sensitized tumor cells to DNA-damaging drugs and thus overcame innate resistance to their action. Combined inhibition of BRCA2 and thymidylate synthase (TS), the enzyme responsible for de novo synthesis of thymidylate (and the source of innate resistance to several treatments), rendered tumor cells responsive to a broader range of drugs and created a state of multi-drug sensitivity. Based on these results, I created a novel BRCA2-targeting antisense oligodeoxynucleotide (ASO) and tested it in the context of cisplatin treatment. ASOs exhibit several advantages over siRNAs in vivo, and some ASO-based drugs have been approved by the FDA. I found that BRCA2 downregulation (with the BRCA2 ASO) enhanced the ability of cisplatin (a cytotoxic, DNA-damaging drug) to control tumor cell proliferation in vitro and metastasis in vivo, and also induced alterations in cellular metabolism. Further studies using the PARP-1 inhibitor olaparib, which is selectively lethal in cells with HRR deficiency, led me to formulate the concept of “reciprocal positive selection for weakness”: in a population heterogeneous for HRR-proficiency, olaparib selects for HRR-proficient cells, while BRCA2 inhibition selects for HRR-deficient cells. Each individual treatment thus selects for cells ‘weak’ to the other in a reciprocal manner, and combined inhibition of both targets should prevent selection-mediated escape. This is a strategy that aims to prevent acquired resistance in a heterogeneous tumor population by nullifying enrichment of specific cell subpopulations. I found that BRCA2 inhibition can render HRR-positive cancer cells (with innate resistance to olaparib) sensitive to PARP inhibition. Furthermore, olaparib monotherapy in a primarily HRR-deficient mixed cell population (3:1 ratio of HRR-deficient:HRR-proficient cells) induced resistance to further olaparib treatment after just one dose. This parallels clinical reports which show that patients with BRCA2-mutated tumors can present with tumors harboring functional BRCA2 protein following olaparib therapy, presumably due to treatment-mediated selection of subclones present at the start of therapy. In my experiments, co-treatment of this same mixed population with BRCA2 ASO and olaparib prevented enrichment based on HRR-proficiency and eliminated the tumor cell population. In addition, treatment of ovarian tumor-bearing mice with BRCA2 siRNA and olaparib in vivo decreased both the number and weight of tumor nodules when compared with each treatment individually. These studies highlight the important role that DNA repair mediators such as BRCA2 play in innate and acquired resistance to treatment, and provide rationale for therapeutic targeting of DNA repair in human tumors

TRYPTOPHAN METABOLISM AND CD8+T-CELL FUNCTION

L-tryptophan is the least common essential amino acid and is necessary for cellular proliferation. In the context of immune regulation, lack of tryptophan in tissue microenvironments and the production of its bioactive metabolites has been implicated as an immunosuppressive phenomenon. The rate limiting enzyme in tryptophan degradation is indoleamine 2,3 dioxygenase (EDO), which metabolizes tryptophan along the L- kynurenine pathway. When induced by interferon signaling, IDO promotes anergy and apoptosis in naive T-cells, and enables the differentiation and activation of T-regulatory cells. However, the role that IDO and tryptophan metabolism play in epitope specific CD8+T-lymphocyte (CTL) function has not yet been investigated. By using a pharmacological inhibitor (1-methyl-D-tryptophan) and knockout (KO) mice independently, IDO was depleted to examine its role in shaping the CTL responses to SV40 large T antigen (TAg) and influenza A virus (IAV). EDO KO and inhibition increased the frequency of CD8+T-cells specific for defined epitopes in both models, as measured by intracellular cytokine staining (ICS). This increase in cell frequency was mediated by a proliferative advantage exhibited by immunodominant EDO KO T-cells when compared with WT counterparts. Bioactive tryptophan metabolites such as L- kynurenine did not play a role in this system, and failed to decrease T-cell responses in either the TAg or IAY model. In addition, IDO KO potentiated the cytotoxic effector function of CTLs in vivo, but did not affect animal morbidity in a disease model of IAV infection

CD5 blockade enhances ex vivo CD8+ T cell activation and tumour cell cytotoxicity

CD5 is expressed on T cells and a subset of B cells (B1a). It can attenuate TCR signalling and impair CTL activation and is a therapeutic targetable tumour antigen expressed on leukemic T and B cells. However, the potential therapeutic effect of functionally blocking CD5 to increase T cell anti‐tumour activity against tumours (including solid tumours) has not been explored. CD5 knockout mice show increased anti‐tumour immunity: reducing CD5 on CTLs may be therapeutically beneficial to enhance the anti‐tumour response. Here, we show that ex vivo administration of a function‐blocking anti‐CD5 MAb to primary mouse CTLs of both tumour‐naïve mice and mice bearing murine 4T1 breast tumour homografts enhanced their capacity to respond to activation by treatment with anti‐CD3/anti‐CD28 MAbs or 4T1 tumour cell lysates. Furthermore, it enhanced TCR signalling (ERK activation) and increased markers of T cell activation, including proliferation, CD69 levels, IFN‐γ production, apoptosis and Fas receptor and Fas ligand levels. Finally, CD5 function‐blocking MAb treatment enhanced the capacity of CD8+ T cells to kill 4T1‐mouse tumour cells in an ex vivo assay. These data support the potential of blockade of CD5 function to enhance T cell‐mediated anti‐tumour immunity

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 invivo 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

Inhibition of BRCA2 and Thymidylate Synthase Creates Multidrug Sensitive Tumor Cells via the Induction of Combined “Complementary Lethality”

A high mutation rate leading to tumor cell heterogeneity is a driver of malignancy in human cancers. Paradoxically, however, genomic instability can also render tumors vulnerable to therapeutic attack. Thus, targeting DNA repair may induce an intolerable level of DNA damage in tumor cells. BRCA2 mediates homologous recombination repair, and BRCA2 polymorphisms increase cancer risk. However, tumors with BRCA2 mutations respond better to chemotherapy and are associated with improved patient prognosis. Thymidylate synthase (TS) is also involved in DNA maintenance and generates cellular thymidylate. We determined that antisense downregulation of BRCA2 synergistically potentiated drugs with mechanisms of action related to BRCA2 function (cisplatin, melphalan), a phenomenon we named “complementary lethality.” TS knockdown induced complementary lethality to TS-targeting drugs (5-FUdR and pemetrexed) but not DNA cross-linking agents. Combined targeting of BRCA2 and TS induced complementary lethality to both DNA-damaging and TS-targeting agents, thus creating multidrug sensitive tumors. In addition, we demonstrated for the first time that simultaneous downregulation of both targets induced combined complementary lethality to multiple mechanistically different drugs in the same cell population. In this study, we propose and define the concept of “complementary lethality” and show that actively targeting BRCA2 and TS is of potential therapeutic benefit in multidrug treatment of human tumors. This work has contributed to the development of a BRCA2-targeting antisense oligdeoxynucleotide (ASO) “BR-1” which we will test in vivo in combination with our TS-targeting ASO “SARI 83” and attempt early clinical trials in the future

IDO Downregulation Induces Sensitivity to Pemetrexed, Gemcitabine, FK866, and Methoxyamine in Human Cancer Cells.

Indoleamine 2,3-dioxygenase-1 (IDO) is an immune regulatory enzyme expressed by most human tumors. IDO levels in tumor cells correlate with increased metastasis and poor patient outcome and IDO is linked to tumor cell resistance to immunotherapy, radiation therapy, and chemotherapy. Knowledge of tumor cell-autonomous effects of IDO, independent of its well-known role in regulating and suppressing anti-tumor immune responses, is limited. Clonal populations of A549 human lung adenocarcinoma cells stably transfected with anti-IDO shRNA or scrambled control shRNA were used to study IDO effects on drug sensitivity and resistance. IFNγ was used to induce IDO in those cells. We show, for the first time, that IDO mediates human tumor cell resistance to the candidate anticancer drugs FK866 (an NAD+ inhibitor), methoxyamine (MX, a base excision repair [BER] inhibitor) and approved anticancer drugs pemetrexed (a folate anti-metabolite) and gemcitabine (a nucleoside analogue), and combined treatment with pemetrexed and MX, in the absence of immune cells. Concurrent knockdown of IDO and thymidylate synthase (TS, a key rate-limiting enzyme in DNA synthesis and repair) sensitizes human lung cancer cells to pemetrexed and 5FUdR to a greater degree than knockdown of either target alone. We conclude that BER in IDO-expressing A549 cells plays a major role in mediating resistance to a range of approved and candidate anticancer drugs. IDO inhibitors are undergoing clinical trials primarily to improve antitumor immune responses. We show that targeting IDO alone or in combination with TS is a potentially valuable therapeutic strategy for cancer treatment, independent of immune activity and in combination with conventional chemotherapy

Preventing and curing citrulline-induced autoimmune arthritis in a humanized mouse model using a Th2-polarizing iNKT cell agonist

Invariant natural killer T (iNKT) cells are innate lymphocytes with unique reactivity to glycolipid antigens bound to non-polymorphic CD1d molecules. They are capable of rapidly releasing pro- and/or anti-inflammatory cytokines and constitute attractive targets for immunotherapy of a wide range of diseases including autoimmune disorders. In this study, we have explored the beneficial effects of OCH, a Th2-polarizing glycolipid agonist of iNKT cells, in a humanized mouse model of rheumatoid arthritis (RA) in which citrullinated human proteins are targeted by autoaggressive immune responses in mice expressing an RA susceptibility human leukocyte antigen (HLA) DR4 molecule. We found for the first time that treatment with OCH both prevents and cures citrulline-induced autoimmune arthritis as evidenced by resolved ankle swelling and reversed histopathological changes associated with arthritis. Also importantly, OCH treatment blocked the arthritogenic capacity of citrullinated antigen-experienced splenocytes without compromising their global responsiveness or altering the proportion of splenic naturally occurring CD4 CD25 FoxP3 regulatory T cells. Interestingly, administering the Th1-promoting iNKT cell glycolipid ligand α-C-galactosylceramide into HLA-DR4 transgenic mice increased the incidence of arthritis in these animals and exacerbated their clinical symptoms, strongly suggesting a role for Th1 responses in the pathogenesis of citrulline-induced arthritis. Therefore, our findings indicate a role for Th1-mediated immunopathology in citrulline-induced arthritis and provide the first evidence that iNKT cell manipulation by Th2-skewing glycolipids may be of therapeutic value in this clinically relevant model, a finding that is potentially translatable to human RA. © 2012 Australasian Society for Immunology Inc

Merger of dynamic two-photon and phosphorescence lifetime microscopy reveals dependence of lymphocyte motility on oxygen in solid and hematological tumors

Abstract Background Low availability of oxygen in tumors contributes to the hostility of the tumor microenvironment toward the immune system. However, the dynamic relationship between local oxygen levels and the immune surveillance of tumors by tumor infiltrating T-lymphocytes (TIL) remains unclear. This situation reflects a methodological difficulty in visualizing oxygen gradients in living tissue in a manner that is suitable for spatiotemporal quantification and contextual correlation with individual cell dynamics tracked by typical fluorescence reporter systems. Methods Here, we devise a regimen for intravital oxygen and cell dynamics co-imaging, termed ‘Fast’ Scanning Two-photon Phosphorescence Lifetime Imaging Microscopy (FaST-PLIM). Using FaST-PLIM, we image the cellular motility of T-lymphocytes in relation to the microscopic distribution of oxygen in mouse models of hematological and solid tumors, namely in bone marrow with or without B-cell acute lymphocytic leukemia (ALL), and in lungs with sarcoma tumors. Results Both in bone marrow leukemia and solid tumor models, TILs encountered regions of varying oxygen concentrations, including regions of hypoxia (defined as pO2 below 5 mmHg), especially in advanced-stage ALL and within solid tumor cores. T cell motility was sustained and weakly correlated with local pO2 above 5 mmHg but it was very slow in pO2 below this level. In solid tumors, this relationship was reflected in slow migration of TIL in tumor cores compared to that in tumor margins. Remarkably, breathing 100% oxygen alleviated tumor core hypoxia and rapidly invigorated the motility of otherwise stalled tumor core TILs. Conclusions This study demonstrates a versatile and highly contextual FaST-PLIM method for phosphorescence lifetime-based oxygen imaging in living animal tumor immunology models. The initial results of this method application to ALL and solid lung tumor models highlight the importance of oxygen supply for the maintenance of intratumoral T cell migration, define a 5 mmHg local oxygen concentration threshold for TIL motility, and demonstrate efficacy of supplementary oxygen breathing in TIL motility enhancement coincident with reduction of tumor hypoxia

Suppression of Immunodominant Antitumor and Antiviral CD8⁺ T Cell Responses by Indoleamine 2,3-Dioxygenase

<div><p>Indoleamine 2,3-dioxygenase (IDO) is a tryptophan-degrading enzyme known to suppress antitumor CD8⁺ T cells (T_CD8). The role of IDO in regulation of antiviral T_CD8 responses is far less clear. In addition, whether IDO controls both immunodominant and subdominant T_CD8 is not fully understood. This is an important question because the dominance status of tumor- and virus-specific T_CD8 may determine their significance in protective immunity and in vaccine design. We evaluated the magnitude and breadth of cross-primed T_CD8 responses to simian virus 40 (SV40) large T antigen as well as primary and recall T_CD8 responses to influenza A virus (IAV) in the absence or presence of IDO. IDO^−/− mice and wild-type mice treated with 1-methyl-D-tryptophan, a pharmacological inhibitor of IDO, exhibited augmented responses to immunodominant epitopes encoded by T antigen and IAV. IDO-mediated suppression of these responses was independent of CD4⁺CD25⁺FoxP3⁺ regulatory T cells, which remained numerically and functionally intact in IDO^−/− mice. Treatment with L-kynurenine failed to inhibit T_CD8 responses, indicating that tryptophan metabolites are not responsible for the suppressive effect of IDO in our models. Immunodominant T antigen-specific T_CD8 from IDO^−/− mice showed increased Ki-67 expression, suggesting that they may have acquired a more vigorous proliferative capacity <i>in vivo</i>. In conclusion, IDO suppresses immunodominant T_CD8 responses to tumor and viral antigens. Our work also demonstrates that systemic primary and recall T_CD8 responses to IAV are controlled by IDO. Inhibition of IDO thus represents an attractive adjuvant strategy in boosting anticancer and antiviral T_CD8 targeting highly immunogenic antigens.</p></div