Immunotherapy has raised high expectations in the treatment of virtually every cancer. Many current efforts are focused on ensuring the efficient delivery of active cytotoxic cells to tumors. It is ...assumed that, once these active cytotoxic cells are correctly engaged to cancer cells, they will unfailingly eliminate the latter, provided that inhibitory factors are in check. T cell bispecific antibodies (TCBs) and chimeric antigen receptors (CARs) offer an opportunity to test this assumption. Using TCB and CARs directed against HER2, here we show that disruption of interferon-gamma signaling confers resistance to killing by active T lymphocytes. The kinase JAK2, which transduces the signal initiated by interferon-gamma, is a component repeatedly disrupted in several independently generated resistant models. Our results unveil a seemingly widespread strategy used by cancer cells to resist clearance by redirected lymphocytes. In addition, they open the possibility that long-term inhibition of interferon-gamma signaling may impair the elimination phase of immunoediting and, thus, promote tumor progression.
Trastuzumab-emtansine (T-DM1) is an antibody-drug conjugate (ADC) approved for the treatment of HER2 (human epidermal growth factor receptor 2)-positive breast cancer. T-DM1 consists of trastuzumab ...covalently linked to the cytotoxic maytansinoid DM1 via a non-cleavable linker. Despite its efficacy, primary or acquired resistance frequently develops, particularly in advanced stages of the disease. Second generation ADCs targeting HER2 are meant to supersede T-DM1 by using a cleavable linker and a more potent payload with a different mechanism of action. To determine the effect of one of these novel ADCs, SYD985, on tumors resistant to T-DM1, we developed several patient-derived models of resistance to T-DM1. Characterization of these models showed that previously described mechanisms-HER2 downmodulation, impairment of lysosomal function and upregulation of drug efflux pumps-account for the resistances observed, arguing that mechanisms of resistance to T-DM1 are limited, and most of them have already been described. Importantly, SYD985 was effective in these models, showing that the resistance to first generation ADCs can be overcome with an improved design.
Abstract
Purpose: Immunotherapy has revolutionized the way several types of cancers are treated, including gastrointestinal tumors. Despite that, most solid tumor patients do not respond or relapse. ...T Cell Bispecific antibodies (TCBs) are a promising immunotherapeutic strategy designed to boost the immune response against tumors, as they redirect cytotoxic T cells against the tumor cells. TCBs are engineered molecules that include binding sites to the T cell receptor and to a tumor-specific or a tumor-associated antigen. Clinical evidence shows that TCBs are an effective immunotherapy to treat cancer. However, little is known about the mechanisms of resistance against this promising therapy. Therefore, there is a need to anticipate the mechanism of response and resistance in order to improve the clinical outcome of patients. All TCBs behave with the same mechanism of action, and the same mechanism of resistance, independent of the target, is expected. In this piece of work, we try to address for the first time this question.
Methods: We use as a tool the gastric HER2+/CEA+ cell line MKN45, and TCBs targeting these antigens. We generated resistant cells against both HER2-TCB (HER2R) and CEACAM5-TCB (CEAR) in vitro. To generate these immunoresistant models we co-cultured the tumor cells with peripheral blood mononuclear cells (PBMCs) and the TCBs for several months in order to obtain resistance. In addition, an in vivo model of resistance against CEACAM5-TCB was also generated with a CEA+ colorectal cancer patient derived xenograft (PDX) in a CD34+ humanized mice model.
Results: We successfully generated resistant models to both TCBs, both in vitro and in vivo. In vitro resistant models were corroborated in a organotypic 3D model as well as in an in vivo humanized PBMC model. We evaluated the cause of resistance, and one plausible mechanism that also happen in hematological malignancies treated with CAR Ts is the loss of the antigen, in this case HER2 and CEA. Contrary to our expectations, we observed a dramatic loss of antigen in the case of CEAR resistant cells, in contrast to HER2R resistant cells, which maintain HER2 levels. In vivo model of resistance against CEACAM5-TCB recapitulate the loss of antigen phenotype.
Conclusions: Our results show for the first time that the mechanism of resistance against TCBs can be totally different depending on the target, and future studies and therapeutic approaches should take this into consideration. In addition, the HER2R resistant cells can be used as a tool to identify unknown mechanisms of resistance against the redirection of T-cells.
Citation Format: Alex Martínez-Sabadell, Enrique J. Arenas, Beatriz Morancho, Irene Rius, Marta Escorihuela, Antonio Luque, Joaquín Arribas. The antigen target is critical in the mechanism of resistance to T-cell based therapies abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1859.
Antibody-drug conjugates (ADC) are antineoplastic agents recently introduced into the antitumor arsenal. T-DM1, a trastuzumab-based ADC that relies on lysosomal processing to release the payload, is ...approved for HER2-positive breast cancer. Next-generation ADCs targeting HER2, such as vic-trastuzumab duocarmazine (SYD985), bear linkers cleavable by lysosomal proteases and membrane-permeable drugs, mediating a bystander effect by which neighboring antigen-negative cells are eliminated. Many antitumor therapies, like DNA-damaging agents or CDK4/6 inhibitors, can induce senescence, a cellular state characterized by stable cell-cycle arrest. Another hallmark of cellular senescence is the enlargement of the lysosomal compartment. Given the relevance of the lysosome to the mechanism of action of ADCs, we hypothesized that therapies that induce senescence would potentiate the efficacy of HER2-targeting ADCs. Treatment with the DNA-damaging agent doxorubicin and CDK4/6 inhibitor induced lysosomal enlargement and senescence in several breast cancer cell lines. While senescence-inducing drugs did not increase the cytotoxic effect of ADCs on target cells, the bystander effect was enhanced when HER2-negative cells were cocultured with HER2-low cells. Knockdown experiments demonstrated the importance of cathepsin B in the enhanced bystander effect, suggesting that cathepsin B mediates linker cleavage. In breast cancer patient-derived xenografts, a combination treatment of CDK4/6 inhibitor and SYD985 showed improved antitumor effects over either treatment alone. These data support the strategy of combining next-generation ADCs targeting HER2 with senescence-inducing therapies for tumors with heterogenous and low HER2 expression.
Combining ADCs against HER2-positive breast cancers with therapies that induce cellular senescence may improve their therapeutic efficacy by facilitating a bystander effect against antigen-negative tumor cells.
Abstract
Introduction: Elimination of cancer cells by effector immune cells represents the culmination of a complex cascade of events, and disruption of any of those events may result in resistance. ...T cell-engaging therapies, such as T cell bispecific antibodies (TCBs) or chimeric antigen receptors (CARs), are raising extraordinary expectations as future treatments for virtually all cancers. Encouraging these expectations, TCBs and CARs have been recently approved to treat some hematologic malignancies. In contrast, TCBs and CARs against solid tumors tested to date, have failed to show clinical efficacy. This failure prompted intense research and the subsequent identification of mechanisms of primary and acquired resistance. Different strategies are being implemented to overcome these mechanisms of resistance. All these mechanisms impinge on the ability of T cells to reach cancer cells and/or on the inhibition of T cells. However, little is known about putative intrinsic mechanisms of resistance of cancer cells. That is, mechanisms deployed by tumor cells to resist killing by fully active and correctly engaged T cells. In this study, we attempted to identify novel intrinsic mechanisms of resistance.
Methods: We have used TCBs and CARs targeting the cell surface receptor HER2 to identify a widespread mechanism of resistance to redirected T cells, using HER2-driven cell lines and Patient-Derived Xenografts (PDX).
Results: We have generated a model of intrinsic resistance to a TCB targeting HER2 by treating during 6 months co-cultures of PBMCs and parental BT474 cells. These resistant cells, named as BT-R, are also resistant to a HER2-CAR, in vitro and in vivo. Using this model, we identified by RNA-seq, a downmodulation of the IFN-gamma signaling pathway. Interestingly, using gain and loss of function approaches, we demonstrated that JAK2 loss is the cause of IFN-gamma deficient response, and as a consequence, resistance to HER2-TCB and CAR-HER2.
Conclusion: We have identified that the kinase JAK2, which transduces the signal initiated by interferon-gamma, is the component preferably disrupted to acquired resistance in all resistant models developed in vitro and in vivo. These results unveil a novel mechanism of resistance to T-cell based therapies, and imply the potential use of JAK2 and IFN-gamma response as a surrogate biomarker of response to immunotherapies. In addition, they open the avenue for the screening for therapies that can overcome deficient interferon-gamma response or restore JAK2 levels, which are promising potential candidates to increase the benefits of immunotherapies.
Citation Format: Enrique Javier Arenas Lahuerta, Alex Martínez-Sabadell, Irene Rius Ruiz, Macarena Román Alonso, Marta Escorihuela, Antonio Luque, Carlos A. Fajardo, Alena Gros, Christian Klein, Joaquín Arribas. JAK2 downmodulation leads to interferon gamma deficient response and resistance to immunotherapy in breast cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1690.
Cholangiocarcinoma (CCA) poses a substantial clinical hurdle as it is often detected at advanced metastatic stages with limited therapeutic options. To enhance our understanding of advanced CCA, it ...is imperative to establish preclinical models that faithfully recapitulate the disease's characteristics. Patient-derived xenograft (PDX) models have emerged as a valuable approach in cancer research, offering an avenue to reproduce and study the genomic, histologic, and molecular features of the original human tumors. By faithfully preserving the heterogeneity, microenvironmental interactions, and drug responses observed in human tumors, PDX models serve as highly relevant and predictive preclinical tools. Here, we present a comprehensive protocol that outlines the step-by-step process of generating and maintaining PDX models using biopsy samples from patients with advanced metastatic CCA. The protocol encompasses crucial aspects such as tissue processing, xenograft transplantation, and subsequent monitoring of the PDX models. By employing this protocol, we aim to establish a robust collection of PDX models that accurately reflect the genomic landscape, histologic diversity, and therapeutic responses observed in advanced CCA, thereby enabling improved translational research, drug development, and personalized treatment strategies for patients facing this challenging disease.
Oxidation of histone H3 at lysine 4 (H3K4ox) is catalyzed by lysyl oxidase homolog 2 (LOXL2). This histone modification is enriched in heterochromatin in triple‐negative breast cancer (TNBC) cells ...and has been linked to the maintenance of compacted chromatin. However, the molecular mechanism underlying this maintenance is still unknown. Here, we show that LOXL2 interacts with RuvB‐Like 1 (RUVBL1), RuvB‐Like 2 (RUVBL2), Actin‐like protein 6A (ACTL6A), and DNA methyltransferase 1associated protein 1 (DMAP1), a complex involved in the incorporation of the histone variant H2A.Z. Our experiments indicate that this interaction and the active form of RUVBL2 are required to maintain LOXL2‐dependent chromatin compaction. Genome‐wide experiments showed that H2A.Z, RUVBL2, and H3K4ox colocalize in heterochromatin regions. In the absence of LOXL2 or RUVBL2, global levels of the heterochromatin histone mark H3K9me3 were strongly reduced, and the ATAC‐seq signal in the H3K9me3 regions was increased. Finally, we observed that the interplay between these series of events is required to maintain H3K4ox‐enriched heterochromatin regions, which in turn is key for maintaining the oncogenic properties of the TNBC cell line tested (MDA‐MB‐231).
T cell bispecific antibodies (TCBs) are engineered molecules that include, within a single entity, binding sites to the T cell receptor and to tumor-associated or tumor-specific antigens. The ...receptor tyrosine kinase HER2 is a tumor-associated antigen in ~25% of breast cancers. TCBs targeting HER2 may result in severe toxicities, likely due to the expression of HER2 in normal epithelia. About 40% of HER2-positive tumors express p95HER2, a carboxyl-terminal fragment of HER2. Using specific antibodies, here, we show that p95HER2 is not expressed in normal tissues. We describe the development of p95HER2-TCB and show that it has a potent antitumor effect on p95HER2-expressing breast primary cancers and brain lesions. In contrast with a TCB targeting HER2, p95HER2-TCB has no effect on nontransformed cells that do not overexpress HER2. These data pave the way for the safe treatment of a subgroup of HER2-positive tumors by targeting a tumor-specific antigen.
Cholangiocarcinoma (CCA) is usually diagnosed at advanced stages, with limited therapeutic options. Preclinical models focused on unresectable metastatic CCA are necessary to develop rational ...treatments. Pathogenic mutations in IDH1/2, ARID1A/B, BAP1, and BRCA1/2 have been identified in 30%-50% of patients with CCA. Several types of tumor cells harboring these mutations exhibit homologous recombination deficiency (HRD) phenotype with enhanced sensitivity to PARP inhibitors (PARPi). However, PARPi treatment has not yet been tested for effectiveness in patient-derived models of advanced CCA.
We have established a collection of patient-derived xenografts from patients with unresectable metastatic CCA (CCA_PDX). The CCA_PDXs were characterized at both histopathologic and genomic levels. We optimized a protocol to generate CCA tumoroids from CCA_PDXs. We tested the effects of PARPis in both CCA tumoroids and CCA_PDXs. Finally, we used the RAD51 assay to evaluate the HRD status of CCA tissues.
This collection of CCA_PDXs recapitulates the histopathologic and molecular features of their original tumors. PARPi treatments inhibited the growth of CCA tumoroids and CCA_PDXs with pathogenic mutations of BRCA2, but not those with mutations of IDH1, ARID1A, or BAP1. In line with these findings, only CCA_PDX and CCA patient biopsy samples with mutations of BRCA2 showed RAD51 scores compatible with HRD.
Our results suggest that patients with advanced CCA with pathogenic mutations of BRCA2, but not those with mutations of IDH1, ARID1A, or BAP1, are likely to benefit from PARPi therapy. This collection of CCA_PDXs provides new opportunities for evaluating drug response and prioritizing clinical trials.
Oncogene-induced senescence (OIS) is a tumor suppressor mechanism. However, senescent cells remain viable and display a distinct secretome (also known as senescence-associated secretory phenotype ...SASP or senescence messaging secretome, SMS) that, paradoxically, includes protumorigenic factors. OIS can be triggered by ectopic overexpression of HER2, a receptor tyrosine kinase and the driving oncogene in a subtype of human breast cancer. However, cellular senescence has not been characterized in HER2-positive tumors.
Using an approach based on their inability to proliferate, we isolated naturally occurring senescent cells from a variety of tumor models including HER2-positive cells, transgenic mice (n = 3), and patient-derived xenografts (PDXs) (n = 6 mice per group from one PDX derived from one patient). Using different biochemical and cell biological techniques, we characterized the secretome of these senescent cells. All statistical tests were two-sided.
We found that senescent cells arise constantly in different models of advanced breast cancers overexpressing HER2 and constitute approximately 5% of tumor cells. In these models, IL-6 and other cytokines were expressed mainly, if not exclusively, by the naturally occurring senescent cells (95.1% and 45.0% of HCC1954 cells and cells from a HER2-positive PDX expressing a senescent marker expressed IL-6, respectively). Furthermore, inhibition of IL-6 impaired the growth of the HER2-positive PDX (mean tumor volume at day 101, control vs anti-huIL-6 treated, 332.2mm(3) 95% confidence interval {CI} = 216.6 to 449.8 vs 114.4mm(3) 95% CI = 12.79 to 216.0, P = .005).
Senescent cells can contribute to the growth of tumors by providing cytokines not expressed by proliferating cells, but required by these to thrive.
PDF
