Drug resistance and new therapies in colorectal cancer

Colorectal cancer (CRC) is often diagnosed at an advanced stage when tumor cell dissemination has taken place. Chemo- and targeted therapies provide only a limited increase of overall survival for these patients. The major reason for clinical outcome finds its origin in therapy resistance. Escape mechanisms to both chemo- and targeted therapy remain the main culprits. Here, we evaluate major resistant mechanisms and elaborate on potential new therapies. Amongst promising therapies is α-amanitin antibody-drug conjugate targeting hemizygous p53 loss. It becomes clear that a dynamic interaction with the tumor microenvironment exists and that this dictates therapeutic outcome. In addition, CRC displays a limited response to checkpoint inhibitors, as only a minority of patients with microsatellite instable high tumors is susceptible. In this review, we highlight new developments with clinical potentials to augment responses to checkpoint inhibitors

Targeting the tumor microenvironment to enhance antitumor immune responses

The identification of tumor-specific antigens and the immune responses directed against them has instigated the development of therapies to enhance antitumor immune responses. Most of these cancer immunotherapies are administered systemically rather than directly to tumors. Nonetheless, numerous studies have demonstrated that intratumoral therapy is an attractive approach, both for immunization and immunomodulation purposes. Injection, recruitment and/or activation of antigen-presenting cells in the tumor nest have been extensively studied as strategies to cross-prime immune responses. Moreover, delivery of stimulatory cytokines, blockade of inhibitory cytokines and immune checkpoint blockade have been explored to restore immunological fitness at the tumor site. These tumor-targeted therapies have the potential to induce systemic immunity without the toxicity that is often associated with systemic treatments. We review the most promising intratumoral immunotherapies, how these affect systemic antitumor immunity such that disseminated tumor cells are eliminated, and which approaches have been proven successful in animal models and patients

Somatic mutation of the cohesin complex subunit confers therapeutic vulnerabilities in cancer

A synthetic lethality-based strategy has been developed to identify therapeutic targets in cancer harboring tumor-suppressor gene mutations, as exemplified by the effectiveness of poly ADP-ribose polymerase (PARP) inhibitors in BRCA1/2-mutated tumors. However, many synthetic lethal interactors are less reliable due to the fact that such genes usually do not perform fundamental or indispensable functions in the cell. Here, we developed an approach to identifying the "essential lethality" arising from these mutated/deleted essential genes, which are largely tolerated in cancer cells due to genetic redundancy. We uncovered the cohesion subunit SA1 as a putative synthetic-essential target in cancers carrying inactivating mutations of its paralog, SA2. In SA2-deficient Ewing sarcoma and bladder cancer, further depletion of SA1 profoundly and specifically suppressed cancer cell proliferation, survival, and tumorigenic potential. Mechanistically, inhibition of SA1 in the SA2-mutated cells led to premature chromatid separation, dramatic extension of mitotic duration, and consequently, lethal failure of cell division. More importantly, depletion of SA1 rendered those SA2-mutated cells more susceptible to DNA damage, especially double-strand breaks (DSBs), due to reduced functionality of DNA repair. Furthermore, inhibition of SA1 sensitized the SA2-deficient cancer cells to PARP inhibitors in vitro and in vivo, providing a potential therapeutic strategy for patients with SA2-deficient tumors

Targeted immunotherapy for HER2-low breast cancer with 17p loss

The clinical challenge for treating HER2 (human epidermal growth factor receptor 2)-low breast cancer is the paucity of actionable drug targets. HER2-targeted therapy often has poor clinical efficacy for this disease due to the low level of HER2 protein on the cancer cell surface. We analyzed breast cancer genomics in the search for potential drug targets. Heterozygous loss of chromosome 17p is one of the most frequent genomic events in breast cancer, and 17p loss involves a massive deletion of genes including the tumor suppressor TP53 Our analyses revealed that 17p loss leads to global gene expression changes and reduced tumor infiltration and cytotoxicity of T cells, resulting in immune evasion during breast tumor progression. The 17p deletion region also includes POLR2A, a gene encoding the catalytic subunit of RNA polymerase II that is essential for cell survival. Therefore, breast cancer cells with heterozygous loss of 17p are extremely sensitive to the inhibition of POLR2A via a specific small-molecule inhibitor, α-amanitin. Here, we demonstrate that α-amanitin-conjugated trastuzumab (T-Ama) potentiated the HER2-targeted therapy and exhibited superior efficacy in treating HER2-low breast cancer with 17p loss. Moreover, treatment with T-Ama induced immunogenic cell death in breast cancer cells and, thereby, delivered greater efficacy in combination with immune checkpoint blockade therapy in preclinical HER2-low breast cancer models. Collectively, 17p loss not only drives breast tumorigenesis but also confers therapeutic vulnerabilities that may be used to develop targeted precision immunotherapy

MAL2 drives immune evasion in breast cancer by suppressing tumor antigen presentation

Immune evasion is a pivotal event in tumor progression. To eliminate human cancer cells, current immune checkpoint therapy is set to boost CD8+ T cell-mediated cytotoxicity. However, this action is eventually dependent on the efficient recognition of tumor-specific antigens via T cell receptors. One primary mechanism by which tumor cells evade immune surveillance is to downregulate their antigen presentation. Little progress has been made toward harnessing potential therapeutic targets for enhancing antigen presentation on the tumor cell. Here, we identified MAL2 as a key player that determines the turnover of the antigen-loaded MHC-I complex and reduces the antigen presentation on tumor cells. MAL2 promotes the endocytosis of tumor antigens via direct interaction with the MHC-I complex and endosome-associated RAB proteins. In preclinical models, depletion of MAL2 in breast tumor cells profoundly enhanced the cytotoxicity of tumor-infiltrating CD8+ T cells and suppressed breast tumor growth, suggesting that MAL2 is a potential therapeutic target for breast cancer immunotherapy

Atractylenolide I enhances responsiveness to immune checkpoint blockade therapy by activating tumor antigen presentation

One of the primary mechanisms of tumor cell immune evasion is the loss of antigenicity, which arises due to lack of immunogenic tumor antigens as well as dysregulation of the antigen processing machinery. In a screen for small-molecule compounds from herbal medicine that potentiate T cell–mediated cytotoxicity, we identified atractylenolide I (ATT-I), which substantially promotes tumor antigen presentation of both human and mouse colorectal cancer (CRC) cells and thereby enhances the cytotoxic response of CD8+ T cells. Cellular thermal shift assay (CETSA) with multiplexed quantitative mass spectrometry identified the proteasome 26S subunit non–ATPase 4 (PSMD4), an essential component of the immunoproteasome complex, as a primary target protein of ATT-I. Binding of ATT-I with PSMD4 augments the antigen-processing activity of immunoproteasome, leading to enhanced MHC-I–mediated antigen presentation on cancer cells. In syngeneic mouse CRC models and human patient–derived CRC organoid models, ATT-I treatment promotes the cytotoxicity of CD8+ T cells and thus profoundly enhances the efficacy of immune checkpoint blockade therapy. Collectively, we show here that targeting the function of immunoproteasome with ATT-I promotes tumor antigen presentation and empowers T cell cytotoxicity, thus elevating the tumor response to immunotherapy

Intratumoral delivery of mRNA : overcoming obstacles for effective immunotherapy

The immunosuppressive tumor microenvironment (TME) is a major obstacle in cancer immunotherapy. Therefore, it has gained attention as a target site. mRNA emerged as a versatile drug class for cancer therapy. We reported that intratumoral administration of mRNA encoding the fusokine F-2 supports tumor-specific T-cell immunity. This study provides proof of concept of the use of mRNA to modulate the TME

Abstract 13: A non-surgical approach to develop an orthotopic colorectal cancer mouse model

Abstract Recapitulating physiologically relevant tumor microenvironment is an important yet a significant challenge in modeling colorectal cancer (CRC) for basic and translational studies. Cecal-wall injection is most widely used for orthotopic animal models of CRC. However, this procedure involves complicated surgical procedures, and its ectotopic nature is a major issue for therapeutic development. Here, we report a non-surgical procedure for establishing orthotopic CRC mouse models, reflecting the appropriate tumor microenvironment to better model the disease development and facilitate translational studies, including drug identification and validation. For this procedure, the mice are fasted overnight. The following day, anesthesia is induced in the study mice (Balb/c or C57BL/6) while placed on a thermostat-controlled heating blanket. Next, a specialized catheter is inserted and distended into the mouse colon to gently block the colon and ensure the localization for tumor development. Hereafter, the colonic epithelium is treated with proteases and mechanical abrasion to cause mild disruption. Following the local gut inflammation, the targeted colon region is incubated with tumor cells, leading to the adhesion of tumor cells (CT26 or MC38) to the colonic epithelium. Based on the animal background and the cell line used, the tumor is developed within 4-6 weeks. Pathological assessment on hematoxylin and eosin stained tumor tissues confirmed the stages of tumor development. Additional adaptations of this procedure include i) tumor development in different mouse strains, ii) local delivery of adeno-Cre in transgenic mice for CRC, and iii) transplantation of tumor organoids in the mouse colon. Overall, our non-surgical procedure overcomes the technical difficulties of surgery and offers several advantages like ease of handling, cost-effectiveness, pain-free with nearly no procedure-associated mortality, and adaptability to several strains. To conclude, this non-surgical procedure is a novel and promising alternative to conventional orthotopic CRC mouse models for translational studies. Citation Format: Samantha Sharma, Kevin Van der Jeught, Zhuolong Zhou, Xinna Zhang, Xiongbin Lu. A non-surgical approach to develop an orthotopic colorectal cancer mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 13

Pharmacokinetics of radiolabeled dimeric sdAbs constructs targeting human CD20

Single-domain antibody fragments (sdAbs) are the smallest functional antigen-binding fragments, derived from heavy chain-only camelid antibodies. When designed as radiolabeled monomeric probes for imaging and therapy of cancer, their fast and specific targeting results in high tumor-to-background ratios early after injection. However, their moderate absolute uptake into tumors might not always be sufficient to treat cancerous lesions. We have evaluated the pharmacokinetics of seven constructs derived from a CD20-targeting monomeric sdAb (alphaCD20). The constructs differed in affinity or avidity towards CD20 (dimeric alphaCD20-alphaCD20 and alphaCD20 fused to a non-targeting control sdAb, referred to as alphaCD20-ctrl) and blood half-lives (alphaCD20 fused to an albumin-targeting sdAb (alphaAlb)=alphaCD20-alphaAlb). The constructs were radiolabeled with (111)In (imaging) and (177)Lu (therapy) using the bifunctional chelator CHX-A"-DTPA and evaluated in vitro and in vivo. In mice, tumor uptake of (177)Lu-DTPA-alphaCD20 decreased from 4.82+/-1.80 to 0.13+/-0.05% IA/g over 72h. Due to its rapid blood clearance, tumor- to-blood (T/B) ratios of >100 were obtained within 24h. Although in vitro internalization indicated that dimeric (177)Lu-DTPA-alphaCD20-alphaCD20 was superior in terms of total cell-associated radioactivity, this was not confirmed in vivo. Blood clearance was slower and absolute tumor uptake became significantly higher for alphaCD20-alphaAlb. Blood levels of (177)Lu-DTPA-alphaCD20-alphaAlb decreased from 68.30+/-10.53 to 3.58+/-0.66% IA/g over 120h, while tumor uptake increased from 6.21+/-0.94 to 24.90+/-2.83% IA/g, resulting in lower T/B ratios. Taken together, these results indicate that the increased size of dimeric alphaCD20-alphaCD20 or the fusion of monomeric alphaCD20 to an albumin- targeting moiety (alphaAlb) counterbalance their improved tumor targeting capacity compared to monomeric alphaCD20