Stroma-mediated Resistance Research Articles

Abstract C035: Spatially limited stroma-mediated resistance potentiates targeted therapy resistance through an integration of multiple juxtacrine and paracrine mechanisms

Abstract Despite the ability to induce strong clinical responses, therapies that target mutational drivers of oncogenic signaling are not curative in advanced cancers, as a subset of cancer cells is capable of surviving therapy and evolving resistance. While therapy resistance is commonly viewed as a cell-intrinsic phenomenon, multiple factors produced by tumors can also confer strong therapy resistance. In contrast to the detailed elucidation of molecular mediators of cell-intrinsic and stroma-mediated (SM) resistance, the relative importance of cell-intrinsic and SM resistance to in vivo therapeutic responses remains poorly defined. To address this gap of knowledge, we used a well-characterized experimental model of ALK+ lung cancer H3122 cell line that exemplifies a duality observed across common models of targetable cancers. Under standard stroma-free in vitro cultures, H3122 cells can survive under therapeutically relevant concentrations of ALK inhibitors (ALKi), eventually acquiring cell-intrinsic therapy resistance. At the same time, co-culture with stromal fibroblasts drastically reduces the sensitivity of H3122 cells to ALKi, indicating the relevance of SM resistance. To understand the impact of SM resistance on in vivo responses, we sought to identify the mechanism(s) responsible for the therapy-protective effects of stromal fibroblasts, subsequently interrogating the impact of the disruption of this mechanism on in vivo therapeutic responses. Our in vitro studies with a large panel of primary stromal fibroblast isolates identified the HGF-cMET axis as the major mechanism responsible for ALKi desensitization. Surprisingly, xenograft validation studies demonstrated a weak effect of HGF-cMET modulation. Histological analyses of tumor tissues revealed that this relative weakness is attributable to a strong, spatially limited HGF-independent component of SM resistance. Mechanistic follow-up demonstrated that the HGF-independent component of SM resistance integrates the effect of multiple well-known juxtacrine and paracrine-acting mechanisms. Whereas the multifactorial nature of stroma-mediated resistance prevented a clear assessment of its relative contribution to in vivo therapy responses, our spatial analyses indicate that SM resistance dominates the ability of tumors to avoid therapeutic elimination. Surprisingly, we found that SM resistance was also a substantial contributor to tumor relapse, indicating that in vivo therapy resistance can integrate both cell-intrinsic and cell-extrinsic effects. This duality of therapy resistance and the multifactorial underpinning of both cell-intrinsic and stroma-mediated resistance present a challenge to therapeutic strategies focused on identifying and targeting specific resistance mechanisms. Our studies suggest that this limitation can be overcome by shifting the therapeutic focus towards shared orthogonal therapeutic sensitivities of tumor cells within residual disease. Citation Format: Bina Desai, Tatiana Miti, Sandhya Prabhakaran, Daria Miroshnychenko, Menkara Henry, Viktoriya Marusyk, Chandler Gatenbee, Marilyn Bui, Jacob Scott, Philipp M. Altrock, Eric Haura, Alexander R.A. Anderson, David Basanta, Andriy Marusyk. Spatially limited stroma-mediated resistance potentiates targeted therapy resistance through an integration of multiple juxtacrine and paracrine mechanisms [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C035.

Dissecting the Spatially Restricted Effects of Microenvironment-Mediated Resistance on Targeted Therapy Responses.

The response of tumors to anti-cancer therapies is defined not only by cell-intrinsic therapy sensitivities but also by local interactions with the tumor microenvironment. Fibroblasts that make tumor stroma have been shown to produce paracrine factors that can strongly reduce the sensitivity of tumor cells to many types of targeted therapies. Moreover, a high stroma/tumor ratio is generally associated with poor survival and reduced therapy responses. However, in contrast to advanced knowledge of the molecular mechanisms responsible for stroma-mediated resistance, its effect on the ability of tumors to escape therapeutic eradication remains poorly understood. To a large extent, this gap of knowledge reflects the challenge of accounting for the spatial aspects of microenvironmental resistance, especially over longer time frames. To address this problem, we integrated spatial inferences of proliferation-death dynamics from an experimental animal model of targeted therapy responses with spatial mathematical modeling. With this approach, we dissected the impact of tumor/stroma distribution, magnitude and distance of stromal effects. While all of the tested parameters affected the ability of tumor cells to resist elimination, spatial patterns of stroma distribution within tumor tissue had a particularly strong impact.

Abstract 1222: Mechanisms of succinate-mediated drug resistance in ER+ breast cancer

Abstract Breast cancer is the most prevalent cancer among women worldwide with estrogen receptor-positive (ER+) cancer constituting 70% of all breast cancers. Although ER+ breast cancers initially respond well to ER-targeted therapies and CDK4/6 inhibitors, approximately 30% patients acquire resistance to targeted therapies. In addition to intrinsic resistance that tumors develop, cancer-associated fibroblasts (CAFs) residing in the tumor microenvironment, also contribute to treatment-resistance and poor survival. The role of secreted metabolites by CAFs has been poorly studied in the context of targeted drug resistance. Therefore, to investigate the mechanism of stroma-mediated resistance to CDK4/6-inhibitors, we propagated patient-derived organoids (PDOs) and CAFs from treatment-naïve and treatment-resistant ER+ breast tumors. Our data show that the CAF conditioned media containing the polar metabolite fraction stimulates resistance to CDK4/6 inhibitors. Therefore, to identify metabolites that drive resistance, we performed an untargeted metabolomics on CAF conditioned media. We observed that the CAF conditioned media was enriched for TCA cycle intermediates, including succinate. Succinate is an oncometabolite that has an essential function in energy metabolism. Aside from causing metabolic reprogramming, succinate can be transported out of the mitochondria to perform other biological processes, such as epigenetic modifications and pseudohypoxia. Furthermore, succinate in the extracellular space binds to the succinate receptor SUCNR1 and induces G-protein coupled receptor-mediated signaling in cells expressing SUCNR1. To assess mechanism of resistance that these metabolites might induce, we treated breast cancer cells with the most enriched metabolites we had identified in our metabolomics assay. These experiments identified succinate as driver of CDK4/6 inhibitor resistance in ER+ breast cancer cells. Furthermore, treating tumor cells with SUCNR1 antagonist increased their sensitivity to CDK4/6-inhibitors, showing that succinate mediated resistance is conveyed via SUCNR1. This suggests that CAF secreted succinate plays a role in supporting a pro-tumor environment by promoting drug resistance towards CDK4/6 inhibitors. Defining this mechanism of resistance will elucidate potential therapeutic targets to augment patient response to therapy. Citation Format: Jenny Hogstrom, Boryana Petrova, Camila Perea, Madelyn Ward, Gerburg Wulf, Laura Collins, Naama Kanarek, Taru Muranen. Mechanisms of succinate-mediated drug resistance in ER+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1222.

Simultaneous isolation of hormone receptor–positive breast cancer organoids and fibroblasts reveals stroma-mediated resistance mechanisms

Recurrent hormone receptor-positive (HR+) breast cancer kills more than 600,000 women annually. Although HR+ breast cancers typically respond well to therapies, approximately 30% of patients relapse. At this stage, the tumors are usually metastatic and incurable. Resistance to therapy, particularly endocrine therapy is typically thought to be tumor intrinsic (e.g., estrogen receptor mutations). However, tumor-extrinsic factors also contribute to resistance. For example, stromal cells, such as cancer-associated fibroblasts (CAFs), residing in the tumor microenvironment, are known to stimulate resistance and disease recurrence. Recurrence in HR+ disease has been difficult to study due to the prolonged clinical course, complex nature of resistance, and lack of appropriate model systems. Existing HR+ models are limited to HR+ cell lines, a few HR+ organoid models, and xenograft models that all lack components of the human stroma. Therefore, there is an urgent need for more clinically relevant models to study the complex nature of recurrent HR+ breast cancer, and the factors contributing to treatment relapse. Here, we present an optimized protocol that allows a high take-rate, and simultaneous propagation of patient-derived organoids (PDOs) and matching CAFs, from primary and metastatic HR+ breast cancers. Our protocol allows for long-term culturing of HR+ PDOs that retain estrogen receptor expression and show responsiveness to hormone therapy. We further show the functional utility of this system by identifying CAF-secreted cytokines, such as growth-regulated oncogene α , as stroma-derived resistance drivers to endocrine therapy in HR+ PDOs.

Comparing the value of mono- vs coculture for high-throughput compound screening in hematological malignancies

AbstractLarge-scale compound screens are a powerful model system for understanding variability of treatment response and discovering druggable tumor vulnerabilities of hematological malignancies. However, as mostly performed in a monoculture of tumor cells, these assays disregard modulatory effects of the in vivo microenvironment. It is an open question whether and to what extent coculture with bone marrow stromal cells could improve the biological relevance of drug testing assays over monoculture. Here, we established a high-throughput platform to measure ex vivo sensitivity of 108 primary blood cancer samples to 50 drugs in monoculture and coculture with bone marrow stromal cells. Stromal coculture conferred resistance to 52% of compounds in chronic lymphocytic leukemia (CLL) and 36% of compounds in acute myeloid leukemia (AML), including chemotherapeutics, B–cell receptor inhibitors, proteasome inhibitors, and Bromodomain and extraterminal domain inhibitors. Only the JAK inhibitors ruxolitinib and tofacitinib exhibited increased efficacy in AML and CLL stromal coculture. We further confirmed the importance of JAK-STAT signaling for stroma-mediated resistance by showing that stromal cells induce phosphorylation of STAT3 in CLL cells. We genetically characterized the 108 cancer samples and found that drug-gene associations strongly correlated between monoculture and coculture. However, effect sizes were lower in coculture, with more drug-gene associations detected in monoculture than in coculture. Our results justify a 2-step strategy for drug perturbation testing, with large-scale screening performed in monoculture, followed by focused evaluation of potential stroma-mediated resistances in coculture.

Abstract 3462: Modeling drug resistance in hormone receptor positive breast cancer using patient derived organoid cultures and cancer associated fibroblasts

Abstract Breast cancer is the most prevalent cancer among women worldwide. Estrogen receptor-positive (ER+) breast cancer constitutes roughly 70% of all breast cancers. Early-stage ER+ breast cancers usually respond well to ER-targeted therapies. However, only 20-40% of patients with advanced ER+ breast cancer respond to ER-targeted therapy. Recently, three inhibitors for the cyclin-dependent kinase 4/6 (CDK4/6) were approved as first-line and/or second-line treatment in combination with ER-targeted therapies. Unfortunately, despite initial response to the combination treatment, patients acquire resistance to both ER-targeted therapies and CDK4/6 inhibitors over time. As a result of this, the 5-year survival of women with metastatic breast cancer is 28%. A large contribution to this poor survival rate is the desmoplastic nature of the tumor microenvironment. Desmoplasia is characterized by the abnormal growth of fibroblasts and a dense extracellular matrix. The role of desmoplasia in breast cancer and its contribution to promoting a pro-tumor environment by limiting nutrients and oxygen has been extensively studied. However, the role of secreted factors such as cytokines and metabolites by cancer-associated fibroblasts (CAFs) has been poorly studied in the context of targeted drug resistance and its effects on tumor behavior. Therefore, to investigate the mechanism of stroma-mediated resistance to targeted therapies, we have propagated patient-derived organoids and CAFs from treatment-naïve and treatment-resistant ER+ breast tumors. Our data shows that conditioned media from CAFs have variable ability to drive resistance to hormone therapy and CDK4/6 inhibitors. To identify secreted factors that drive resistance, we have performed cytokine arrays and untargeted metabolomics of the CAF lines that were the strongest drivers of resistance. We have identified several cytokines, such as IL-5, IL-8 and YKL40, that were secreted by all CAF lines. Furthermore, many CAF lines secreted the TCA cycle intermediates α-ketoglutarate and succinate, which have been implicated as oncometabolites in solid tumors. Our preliminary data suggests that YKL40, and succinate induce drug resistance. Together, this suggests that these factors secreted by CAFs play a role in supporting a pro-tumor environment by promoting drug resistance. Defining this mechanism of resistance to ER-targeted therapy and CDK4/6 inhibitors will elucidate potential therapeutic targets to enhance patient response to therapy. Citation Format: JENNY HOGSTROM, Kayla Cruz, Jaymin Patel, Tejas Mehta, Angelica Warren, Taru Muranen. Modeling drug resistance in hormone receptor positive breast cancer using patient derived organoid cultures and cancer associated fibroblasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3462.

ERK1/2 Inhibition Overcomes Resistance in Acute Myeloid Leukemia (AML) and Alters Mitochondrial Dynamics

Background: Presence at diagnosis or acquisition of activating RAS pathway mutations is a pervasive mechanism of resistance to therapy in AML. Efforts to directly target mutant RAS have been unsuccessful and the efficacy of BRAF and MEK inhibitors has been limited due to compensatory reactivation of MAPK signaling. ERK1/2 (ERK) is a key downstream component in the MAPK pathway and therefore represents an attractive target for inhibiting MAPK signaling. Compound 27 (1) is a dual-mechanism inhibitor of ERK that inhibits both the catalytic activity of ERK and its phosphorylation by MEK. It is a close analog of ASTX029, a dual-mechanism ERK inhibitor currently under clinical investigation in solid tumors (NCT03520075).Objectives: We analysed the preclinical activity of Compound 27 in AML, investigated its mechanism of action and ability to overcome resistance.Results: Using a panel of 9 AML cell lines, the IC50 value for single agent Compound 27 was in the low to intermediate nanomolar range (1.89-388 nM). Decreased ERK phosphorylation was confirmed by Western blot analysis. To better characterize the biological effects of Compound 27, we performed mass cytometry (CyTOF) analysis of NRAS-mutated OCI-AML3 cells. This experiment showed approximately 75% downregulation of CyclinB1 and cMyc in 250 nM drug-treated cells versus untreated cells (Figure 1a). The expression of anti-apoptotic proteins, including MCL1, BclXL and Bcl2, were also decreased. Western blot analysis confirmed increased cleaved PARP, and reduced cMyc and cell cycle-related proteins CyclinB1, CyclinD1 and CDK4 with Compound 27 treatment. In isogenic cells, p53 knock-down had no effect on the efficacy of Compound 27.We next investigated the efficacy of simultaneous inhibition of ERK and Bcl-2 in AML cells. Compound 27 sensitized OCI-AML3 cells, which are intrinsically resistant to ABT-199 (a Bcl-2 inhibitor), to treatment with ABT-199 and shifted the cytostatic effect of the single agents to a cytotoxic effect with a combination index (CI) of 0.008 (cell death 91% for combination versus 20% with ABT-199 alone). This suggests strong synergistic effects of combination treatment (Figure 1b). In OCI-AML2 cells with acquired resistance to ABT-199, the combination increased apoptosis to 80% as compared to 20% with ABT-199 alone. Compound 27 sensitized bulk CD45+ as well as CD34+CD38-leukemia progenitor cells to ABT-199. Compound 27 also sensitized FLT3-ITD mutant human AML cell lines MOLM13, MOLM14, MV-4-11 and murine Ba/F3-ITD cells to the FLT3 inhibitor AC220 (CI in MOLM13=0.3). Synergy of Compound 27 and 5-azacitidine was also observed (p=0.009).Leukemia microenvironment-mediated resistance to therapy is partly mediated by MAPK activation. We co-cultured OCI-AML3 and MOLM13 cells with normal bone marrow-derived mesenchymal stromal cells (NMSCs) to mimic the bone marrow microenvironment and analysed the effect of Compound 27 in combination with either ABT-199 or AC220. Combination drug treatment were more effective in terms of cytoreduction and apoptosis induction in coculture. However, neither combination was able to completely overcome stroma-mediated resistance (Figure 1b). Analysis of other stroma-relevant molecules in coculture showed that CXCR4 was increased while CD44 was decreased in response to ERK inhibition. Effective reactive oxygen species (ROS) mitigation and hyper-active mitochondrial fission is important for maintaining “stemness” of AML cells (2). ERK phosphorylates DRP1, which is necessary for mitochondrial fission. Treatment of OCI-AML3 cells with Compound 27 led to increased mitochondrial ROS, decreased levels of pDRP1(Ser616) and increased mitochondrial length, suggesting impaired fission and reduced “stemness” of AML cells (Figure 1c).Conclusion: ERK inhibition by Compound 27 synergizes with 5-azacitidine, ABT-199 and AC220 and can overcome primary or acquired resistance. The impact on mitochondrial dynamics suggests a potential impact on leukemia stem cells. Additional mechanistic confirmatory work is in progress.

Stroma-Mediated Resistance to S63845 and Venetoclax through MCL-1 and BCL-2 Expression Changes Induced by miR-193b-3p and miR-21-5p Dysregulation in Multiple Myeloma.

BH3-mimetics targeting anti-apoptotic proteins such as MCL-1 (S63845) or BCL-2 (venetoclax) are currently being evaluated as effective therapies for the treatment of multiple myeloma (MM). Interleukin 6, produced by mesenchymal stromal cells (MSCs), has been shown to modify the expression of anti-apoptotic proteins and their interaction with the pro-apoptotic BIM protein in MM cells. In this study, we assess the efficacy of S63845 and venetoclax in MM cells in direct co-culture with MSCs derived from MM patients (pMSCs) to identify additional mechanisms involved in the stroma-induced resistance to these agents. MicroRNAs miR-193b-3p and miR-21-5p emerged among the top deregulated miRNAs in myeloma cells when directly co-cultured with pMSCs, and we show their contribution to changes in MCL-1 and BCL-2 protein expression and in the activity of S63845 and venetoclax. Additionally, direct contact with pMSCs under S63845 and/or venetoclax treatment modifies myeloma cell dependence on different BCL-2 family anti-apoptotic proteins in relation to BIM, making myeloma cells more dependent on the non-targeted anti-apoptotic protein or BCL-XL. Finally, we show a potent effect of the combination of S63845 and venetoclax even in the presence of pMSCs, which supports this combinatorial approach for the treatment of MM.

CXCR4 Inhibition Enhances Efficacy of FLT3 Inhibitors in FLT3-Mutated AML Augmented by Suppressed TGF-b Signaling.

Given the proven importance of the CXCL12/CXCR4 axis in the stroma–acute myeloid leukemia (AML) interactions and the rapid emergence of resistance to FLT3 inhibitors, we investigated the efficacy and safety of a novel CXCR4 inhibitor, LY2510924, in combination with FLT3 inhibitors in preclinical models of AML with FLT3-ITD mutations (FLT3-ITD-AML). Quizartinib, a potent FLT3 inhibitor, induced apoptosis in FLT3-ITD-AML, while LY2510924 blocked surface CXCR4 without inducing apoptosis. LY2510924 significantly reversed stroma-mediated resistance against quizartinib mainly through the MAPK pathway. In mice with established FLT3-ITD-AML, LY2510924 induced durable mobilization and differentiation of leukemia cells, resulting in enhanced anti-leukemia effects when combined with quizartinib, whereas transient effects were seen on non-leukemic blood cells in immune-competent mice. Sequencing of the transcriptome of the leukemic cells surviving in vivo treatment with quizartinib and LY2510924 revealed that genes related to TGF-β signaling may confer resistance against the drug combination. In co-culture experiments of FLT3-ITD-AML and stromal cells, both silencing of TGF-β in stromal cells or TGF-β-receptor kinase inhibitor enhanced apoptosis by combined treatment. Disruption of the CXCL12/CXCR4 axis in FLT3-ITD-AML by LY2510924 and its negligible effects on normal immunocytes could safely enhance the potency of quizartinib, which may be further improved by blockade of TGF-β signaling.

Combination of a mitogen-activated protein kinase inhibitor with the tyrosine kinase inhibitor pacritinib combats cell adhesion-based residual disease and prevents re-expansion of FLT3-ITD acute myeloid leukaemia.

Minimal residual disease (MRD) in acute myeloid leukaemia (AML) poses a major challenge due to drug insensitivity and high risk of relapse. Intensification of chemotherapy and stem cell transplantation are often pivoted on MRD status. Relapse rates are high even with the integration of first-generation FMS-like tyrosine kinase 3 (FLT3) inhibitors in pre- and post-transplant regimes and as maintenance in FLT3-mutated AML. Pre-clinical progress is hampered by the lack of suitable modelling of residual disease and post-therapy relapse. In the present study, we investigated the nature of pro-survival signalling in primary residual tyrosine kinase inhibitor (TKI)-treated AML cells adherent to stroma and further determined their drug sensitivity in order to inform rational future therapy combinations. Using a primary human leukaemia-human stroma model to mimic the cell-cell interactions occurring in patients, the ability of several TKIs in clinical use, to abrogate stroma-driven leukaemic signalling was determined, and a synergistic combination with a mitogen-activated protein kinase (MEK) inhibitor identified for potential therapeutic application in the MRD setting. The findings reveal a common mechanism of stroma-mediated resistance that may be independent of mutational status but can be targeted through rational drug design, to eradicate MRD and reduce treatment-related toxicity.

IMiDs mobilize acute myeloid leukemia blasts to peripheral blood through downregulation of CXCR4 but fail to potentiate AraC/Idarubicin activity in preclinical models of non del5q/5q- AML

ABSTRACTTreatment for acute myeloid leukemia (AML) remains suboptimal and many patients remain refractory or relapse upon standard chemotherapy based on nucleoside analogs plus anthracyclines. The crosstalk between AML cells and the BM stroma is a major mechanism underlying therapy resistance in AML. Lenalidomide and pomalidomide, a new generation immunomodulatory drugs (IMiDs), possess pleiotropic anti-leukemic properties including potent immune-modulating effects and are commonly used in hematological malignances associated with intrinsic dysfunctional BM such as myelodysplastic syndromes and multiple myeloma. Whether IMiDs may improve the efficacy of current standard treatment in AML remains understudied. Here, we have exploited in vitro and in vivo preclinical AML models to analyze whether IMiDs potentiate the efficacy of AraC/Idarubicin-based standard AML chemotherapy by interfering with the BM stroma-mediated chemoresistance. We report that IMiDs do not exert cytotoxic effects on either non-del5q/5q- AML cells nor BM-MSCs, but they enhance the immunomodulatory properties of BM-MSCs. When combined with AraC/Idarubicin, IMiDs fail to circumvent BM stroma-mediated resistance of non-del5q/5q- AML cells in vitro and in vivo but induce robust extramedullary mobilization of AML cells. When administered as a single agent, lenalidomide specifically mobilizes non-del5q/5q- AML cells, but not healthy CD34+ cells, to peripheral blood (PB) through specific downregulation of CXCR4 in AML blasts. Global gene expression profiling supports a migratory/mobilization gene signature in lenalidomide-treated non-del5q/5q- AML blasts but not in CD34+ cells. Collectively, IMiDs mobilize non-del5q/5q- AML blasts to PB through CXCR4 downregulation, but fail to potentiate AraC/Idarubicin activity in preclinical models of non-del5q/5q- AML.

The Bone Marrow Environment Promotes Resistance to Mitoxantrone and Etoposide By Distinct Mechanisms in Pediatric AML

Despite aggressive treatments, death from chemoresistant disease still occurs for almost half of children with AML. One possible mechanism of chemoresistance is enhanced DNA damage repair. Mitoxantrone and etoposide are standard chemotherapy for AML, both leading to DNA damage by inhibition of topoisomerase II. Homologous recombination (HR) and non-homologous end joining (NHEJ) are the two main processes for DNA damage repair, with ataxia-telangiectaxia mutated (ATM) kinase and DNA-dependent protein kinase (DNA-PK) as key components, respectively. Both kinases phosphorylate histone H2AX (gH2AX), which facilitates DNA damage repair. Additionally, the bone marrow stromal environment protects a subset of cells from chemotherapy, but the mechanisms of resistance remain unknown. To study leukemia-stroma interactions, we used HS5 and HS27A human bone marrow stromal cells. In co-culture studies, we found that stroma-mediated resistance to mitoxantrone was mediated by both stromal soluble factors and cell-cell contact, whereas resistance to etoposide mainly by physical contact with stroma. Further, we recently reported that stromal CYR61 promotes resistance to mitoxantrone, but not etoposide (Long, et al, 2015, Br J Haematol, 170:704).To further study the mechanism underlying stroma-induced chemotherapy resistance, 44 diagnostic AML patient samples from the Children’s Oncology Group were co-cultured on stromal cells, or cultured alone. The samples were treated with 100 nM mitoxantrone (n=27) or 10 µM etoposide (n=32) for 24h. Fifteen samples had sufficient cells for both chemotherapy treatments. Cells were analyzed by FACS, and stromal cells, which express mOrange, and lymphocytes (CD45high, SSClow) were excluded. We measured intracellular levels of cleaved PARP (cPARP) as an apoptosis marker, and gH2AX as a DNA damage signaling marker. As expected, AML cell viability (%cPARP-) after etoposide treatment was significantly higher in the stromal co-cultures (61.2 ±3.3% for AML cells cultured alone, v. 83.2 ±1.8% in HS5 co-cultures, p<0.0001). Results were similar for mitoxantrone treatment. We also found increased DNA damage signaling (%cPARP-/gH2AX+) after mitoxantrone treatment for AML cells in stromal co-culture (34.5 ±3.0% in cells cultured alone, v. 58.6 ±3.1% in HS5 co-cultures, p<0.0001). However, DNA damage signaling was not significantly increased after etoposide treatment. For 5 samples treated with both chemotherapy agents, we also measured pDNA-PKcs and pATM by FACS. Treatment with mitoxantrone, but not etoposide, induced more pDNA-PKcs (MFI, 25.7 ±4.2 untreated, v. 61.7 ±5.1 mitoxantrone, p<0.001, v. 31.5 ±4.8 etoposide) in AML cells cultured alone. However, stroma did not further increase pDNA-PKcs. These results suggest that the NHEJ pathway is important for the repair of DNA damage caused by mitoxantrone, and that stromal cells increase DNA damage signaling by a mechanism not involving increased pDNA-PKcs.To better understand etoposide resistance, 29 of the 44 primary AML samples were cultured on stroma overnight, and activation of intracellular signaling pathways, including pY-STAT3, pY-STAT5, and pERK1/2, was measured by FACS. Responses were heterogeneous overall, but we found several patterns of stroma-induced signaling. For example, a sample that strongly activated a given pathway when co-cultured with HS5 also responded strongly to HS27A cells, and this was particularly true for pY-STAT3 (R=0.71, p<0.0001) and pY-STAT5 (R=0.76, p<0.0001). We also found a correlation between pY-STAT3 and pY-STAT5, such that samples that showed a strong activation of STAT3 with stromal co-culture had a similar activation of STAT5 (R=0.94, p<0.0001, on HS27A). As for signaling pathways in relationship to apoptosis, higher levels of pERK1/2 were associated with lower levels of apoptosis (R=-0.5182, p<0.01, on HS27A), suggesting that ERK1/2 activation may promote resistance to etoposide.In summary, we found that DNA damage signaling is induced in AML cells by mitoxantrone, and it is augmented in cells co-cultured with stroma, likely contributing to mitoxantrone resistance. This mechanism does not occur with etoposide. Instead, we found that stromal environment-induced ERK1/2 signaling may enhance etoposide resistance in pediatric AML patient samples. Further studies to confirm the role of pERK1/2 in stroma-induced etoposide resistance are underway. DisclosuresNo relevant conflicts of interest to declare.

ARV-825, a BRD4 Inhibitor, Leads to Sustained Degradation of BRD4 with Broad Activity Against Acute Myeloid Leukemia and Overcomes Stroma Mediated Resistance by Modulating Chemokine Receptor, Cell Adhesion and Metabolic Targets

ARV-825, a BRD4 Inhibitor, Leads to Sustained Degradation of BRD4 with Broad Activity Against Acute Myeloid Leukemia and Overcomes Stroma Mediated Resistance by Modulating Chemokine Receptor, Cell Adhesion and Metabolic Targets

Abstract A45: Exosome transfer from stromal to breast cancer cells regulates therapy resistance pathways in triple-negative breast cancer

Abstract Breast cancer cells can achieve protection from DNA damage both through cell-autonomous mechanisms and intercellular communication with the tumor microenvironment. Previously, we described an Interferon-Related DNA Damage Resistance Signature comprising of a network of interferon-stimulated genes (ISGs) that promotes and clinically predicts chemotherapy and radiation resistance in breast cancer. Here, we examine the heterotypic tumor-stroma interactions that regulate ISGs. We show that STAT1 and other ISGs are induced in breast cancer cells following interaction with stroma. STAT1 induces NOTCH3 expression, explaining a requirement for juxtacrine signaling. Moreover, we report that stroma upregulates ISG expression in breast cancer cells through exosomes. Exosomes transferred from the stroma and ISG induction are both dependent on RAB27B. In mice, targeting these pathways abrogate stroma-mediated resistance and results in long-term tumor-free survival. Analysis of primary human tumors supports the role of anti-viral/NOTCH3 pathways in NOTCH signaling and stroma-mediated resistance. To determine whether these observations can further predict clinical efficacy, cell lines derived from a genetically engineered mouse model for p53-induced breast cancer, K14cre;Brca1F/F;p53F/F, were tested. We show that stroma induces breast cancer ISGs through exosomes, which in turn can induce signaling to activate NOTCH3 and regulate DNA damage resistance. Citation Format: Tony J. Wu, Barzin Y. Nabet, Bihui Xu, Mirjam C. Boelens, Jos Jonkers, Andy J. Minn. Exosome transfer from stromal to breast cancer cells regulates therapy resistance pathways in triple-negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A45.

Abstract 927: Fibroblast-induced switching to the invasive phenotype and PI3K-mTOR signaling protects melanoma cells from BRAF inhibitors

Abstract Phenotypic heterogeneity of cancer cells can reason diversity in therapy responses within the same tumor, which might influence the overall efficacy of the treatment. Tumor stroma is an important contributor to intratumoral heterogeneity and can play a significant modulatory role in therapy response/resistance. Through studies on biological mechanisms of stroma-promoted resistance, novel targets could be identified for combination therapies aimed to eradicate both stroma-dependent and independent counterparts of the tumor. In this study we explore how the efficacy of the BRAF inhibitor (BRAFi) vemurafenib, a targeted agent commonly used against BRAF-mutant malignant melanoma, is modulated by stromal cells. By using multiple co-culture systems and experimental metastasis models, we showed that in the presence of lung fibroblasts, adjacent melanoma cells respond poorly to BRAFi. The protective influence of stroma was associated with stroma-induced changes in the melanoma cell phenotype, which was mapped by global gene expression and proteome analysis. We revealed that under the influence of fibroblasts, melanoma cells underwent a phenotype transition to the invasive, mesenchymal-like state characterized by down-regulation of melanocytic markers (MITF and its targets), up-regulation of receptor tyrosine kinases (RTKs)/RTK-linked signaling (like AXL or PDGFR activating down-stream PI3K) and elevation of extracellular-matrix fibronectin. We propose that these alterations allow melanoma cells to utilize alternative signaling pathways, like RTK-PI3K-mTOR instead of BRAF-driven MAPK, which reduces sensitivity to BRAFi. This scenario is further supported by the observations that: i) upon BRAFi treatment, stroma-protected melanoma maintained high levels of phospho-ribosomal protein S6 (pS6), a mTOR effector protein; ii) inhibition of mTOR or the upstream pathway PI3K together with BRAF eradicated pS6high subpopulations and enhanced the anti-proliferative effect in stroma-interacting melanoma; iii) the benefit of mTOR and BRAF co-inhibition was also seen in early-stage lung metastases in vivo. In conclusion, our findings signify the importance of stromal cells, specifically lung fibroblasts, in regulating melanoma cell phenotype and signaling, which impairs response to BRAFi. The stroma-induced invasive phenotype determinants that facilitate RTK-PI3K-mTOR signaling (e.g. AXL, PDGFR or fibronectin-binding integrins) could represent potential targets for overcoming stroma-mediated resistance to BRAFi. Currently, we are testing BRAFi in combination with several novel inhibitors of the identified RTKs and performing a cancer drug sensitivity screen to reveal the most effective drug combinations against stroma-interacting melanoma cells. Citation Format: Kotryna Seip, Karianne Giller Fleten, Anna Barkovskaya, Vigdis Nygaard, Mads Haugen Haugen, Birgit Øvstebø Engesæter, Gunhild Mari Mælandsmo, Lina Prasmickaite. Fibroblast-induced switching to the invasive phenotype and PI3K-mTOR signaling protects melanoma cells from BRAF inhibitors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 927.

Microenvironmental Interactions up-Regulate CD20 Expression in CLL B Cells through the CXCR4/SDF-1 Axis: Implications for CD20-Targeting Antibodies and the Use of BCR-Inhibitors in Combination

Abstract The aim of this study was to investigate the role of microenvironmental interactions in the regulation of CD20 expression, since down-modulation of CD20 in the context of immune niches would be a straightforward explanation for the stroma-mediated rituximab resistance described by us and others (Mraz et al., BJH, 2011; Buchner et al., BJH, 2010; Marquez et al., BJH, 2015). Firstly, we co-cultured primary CLL cells with the bone marrow stromal cell line HS-5 for 24/48/72 hrs and analyzed CD20 surface expression by flow cytometry. Suprisingly, CD20 levels were significantly up-regulated (P=0.03) on CLL cells co-cultured with HS-5 in comparison to CLL cells cultured on plastic. The changes in CD20 expression levels are likely to play a direct role in microenvironmental interactions and especially BCR signaling in immune niches (Uchida et al., Int Immunol, 2004). To test this hypothesis we assessed the CD20 expression on CLL cell populations defined according to CXCR4 and CD5 levels. The low-level surface expression of chemokine receptor CXCR4 and high-level expression of activation molecule CD5 can characterize B cells that have recently exited the lymph nodes (Calissano et al., Mol Med, 2011). CXCR4dim CD5bright cells had ~2-fold higher surface CD20 expression when compared to CXCR4bright CD5dim CLL cells circulating in the blood of the same patient for a longer time (P&lt;0.0001, N=21) and CD20 expression clearly decreased with the transition of CLL cells from CXCR4dim to CXCR4bright together with the decrease of CD5 expression. Further, sorted CXCR4dim CD5bright CLL cells had also ~2 times higher CD20 mRNA expression (P=0.002, N=8) suggesting that changes in CD20 expression were due to changes in its mRNA levels rather than a surface modulation. This led us to hypothesize that CXCR4/SDF-1 is directly implicated in CD20 regulation. Indeed, CLL cells treated with SDF-1α (CXCL12), a ligand for CXCR4 which is produced by HS-5, up-regulated surface CD20 (P=0.03). We also observed that CLL cells with higher expression of CD20 had higher levels of surface Ig (P=0.0015, N=12) and this was coupled with higher responsiveness to BCR crosslinking with anti-IgM as measured by calcium flux (P=0.005). Therefore, we investigated the effect of ibrutinib on CD20 levels as ibrutinib is a BCR-signaling pathway inhibitor and prevents CLL cells from responding to microenvironmental stimuli, including chemokine signaling. We observed that treatment of CLL cells with ibrutinib in vitro resulted in CD20 down-modulation (P&lt;0.001). The levels of CD20 in CLL cells were also strongly down-modulated in samples obtained before vs. during ibrutinib treatment of CLL patients in vivo (pre- ibrutinib vs. day 15 and week 5/12, N=8; P=0.02, P=0.01, P=0.07, respectively). These data suggest that a reduction of the target antigen (CD20) is not the cause for rituximab resistance in the context of immune niches, and the sustained activity of ibrutinib and rituximab combinations in clinical trials (Burger et al., Lancet Oncol, 2014) suggests that ibrutinib has other mechanisms that allow for rituximab efficacy. We focused on the regulation of anti-apoptotic molecules, namely Mcl1, since that was shown to protect from rituximab-induced apoptosis and to be rapidly down-modulated by rituximab infusion in vivo (Byrd et al., Blood, 2002; Hussain et al., Clin Cancer Res, 2007). Indeed, we observed higher levels of Mcl1 mRNA in the CXCR4dim CD5bright CLL subpopulation (N=8, P=0.04), and in CLL cells co-cultured with stromal cells. Importantly, Mcl1 was down-regulated after ibrutinib treatment both in vitro and in ibrutinib treated patients. Conclusion: Microenvironmental interactions up-regulate CD20 expression in CLL B cells through the CXCR4/SDF-1 axis, and the ibrutinib-mediated impairment of microenvironmental interactions down-regulates CD20 expression. This study reveals a novel regulation of CD20 levels in the context of immune niches, which has important implications for CD20-targeting antibodies and the use of BCR-inhibitors in combination. Supported by: SoMoPro II-no.4SGA8684 (cofinanced by the European Union and the South-Moravian Region); NGS-PTL(306242); EHA Fellowship award; IGA MZ CR NT11218-6/2010; MUNI/A/1180/2014; CZ.1.05/1.1.00/02.0068; G.P. is a city of Ostrava scholarship holder. #G.P. and V.S. contributed equally to this study. contact: marek.mraz@email.cz Disclosures Mayer: Janssen: Research Funding. Davids:Genentech: Other: ad board; Pharmacyclics: Consultancy; Janssen: Consultancy.

Mapping Microenvironment-Mediated Signaling Dependency of Targeted Inhibitors: A Mechanism-Based Approach of Selecting Effective Therapy Targeting Drug Resistant AML

Acute myeloid leukemia (AML) has a high relapse rate, with drug resistance often observed in relapsed patients. The leukemic microenvironment shelters surviving AML cells following initial treatment, and facilitates the development of drug resistance in AML that accompanies disease relapse (Konopleva et al., JCO 2011). Activation of PI3K/AKT/mTOR, anti-apoptotic Bcl-2 and MDM2/p53 signaling have all been detected in relapsed AML, and were reported as resistance mechanisms mediated by stromal cells in the leukemia microenvironment. Temsirolimus, ABT737 and Nutlin3a are small molecule inhibitors that block key molecules (mTOR, Bcl-2 and MDM2) in their respective pathways; each demonstrated therapeutic efficacy in AML treatment (Zeng, et al., Blood 2007; Konopleva et al., Cancer Cell 2006; Kojima et al., Blood 2011).In this study, we investigated stroma-mediated drug-resistance mechanisms to these target inhibitors, aiming to identify effective combinations to eradicate residual AML. Using reverse phase-protein array (RPPA) technology we profiled 53 key molecules in 11 signaling pathways (Figure 1) in 20 primary AML samples that were treated with temsirolimus, ABT-737, or Nutlin3a alone; or the combination of temsirolimus plus ABT-737 or Nutlin3a under mono-culture and stroma co-culture conditions. Stroma-mediated mechanisms of resistance were identified by comparing the signaling network alterations triggered by single inhibitor or dual inhibitors under the different culture conditions.Our results indicate that alterations in the stroma-regulated network responsible for cell surivial are specific to each inhibitor. Stroma upregulated p53-independent MDM2 expression (14/20 samples) and activated p-BAD Ser136 (9/20) in samples treated with temsirolimus; activated p-AKT Ser473 (14/20), p-PRAS40 Thr246 (10/20), increased expression of Foxo3A (10/20), and upregulated p53-independent MDM2 (12/20) in samples treated with ABT-737; it activated p-AKT Ser473 (13/20) and upregulated p53-independent MDM2 expression (14/20) in samples treated with Nultin3a. The finding that activation of the PI3K/AKT/mTOR pathway was a common survival mechanism mediated by stroma in response to Bcl-2 and MDM2 inhibition provided the rationale for combination strategies using temsirolimus plus ABT-737 or Nutlin3a. Mapping of the signaling network revealed that the number of affected targets and pathways were significantly increased in the co-cultured samples treated with dual inhibitors: temsirolimus plus ABT-737 (15 targets in 7 pathways), temsirolimus plus Nutlin3a (13 targets in 5 pathways), as compared to those treated with single agent: temsirolimus (7 targets in 3 pathways), ABT-737 (6 targets in 2 pathways) and Nutlin3a (2 targets in 2 pathways). Network analysis revealed that temsirolimus plus ABT-737 blocked up- and downstream targets within the PI3K/AKT/mTOR pathway (#1) (PDK1 vs. 4EBP1), and simultaneously inhibited targets in multiple pro-survival pathways: AKT/mTOR downstream targets (#2), STAT3 (#4), IAP family (#8), p53 family (#7), Wnt-beta Catenin (#6), Bcl-2 family (#5). Similarly, temsirolimus and Nutlin3a blocked the targets within the PI3K/AKT/mTOR signaling (#1) (AKT, mTOR, p-4EPB1, p-S6), and the targets in pro-survival pathways such as targets downstream of AKT/mTOR (#2), cell cycle regulation (#9), p53 family (#7), Wnt-beta Catenin (#6). Apoptosis was measured in 11 of 20 samples under co-culture condition; both combinations showed increased apoptosis induction: temsirolimus (mean± SE) 8.5 ± 2.7%, ABT737 21.1± 2 %, temsirolimus plus ABT737 37 ± 2.7% (n =11); temsirolimus 13.9 ± 3.6%, Nutlin3a 16.9 ± 5.4%, temsirolimus plus Nutlin3a 35.7 ± 2.8% (n=6).Altogether, using proteomic profiling we identified stroma-mediated resistance mechanisms to target-specific inhibitors, and examined the effects of combined blockade of PI3K/AKT/mTOR signaling with Bcl-2 and MDM2 inhibition at the molecular level. We demonstrated that the mechanism-based selection of combined inhibition is able to abrogate stroma-mediated drug-resistance and facilitate cell death. This approach offers the important guidance for clinical drug selection and tailoring treatment regimens to eliminate resistant AML. [Display omitted] DisclosuresKonopleva:Novartis: Research Funding; AbbVie: Research Funding; Stemline: Research Funding; Calithera: Research Funding; Threshold: Research Funding.

Focal Adhesion Kinase Inhibitors Reverse the Stromal Adhesion Phenotype of Ikaros-Mutant B-ALL, Induce Apopotosis, and Synergize with ABL1 Tyrosine Kinase Inhibitors: A New Paradigm for Pathogenesis and Therapy of High-Risk B-ALL

B-cell acute lymphoblastic leukemia (B-ALL) is a malignancy of precursor B-lymphocytes affecting both children and adults. Deletions and dominant-negative mutations in IKZF1, the gene encoding the Ikaros transcription factor, are found in ~85% of Ph+ B-ALL and in some cases of Ph– B-ALL, and are associated with poor prognosis. Genomic studies of high-risk Ph– or “Ph-like” B-ALLs have revealed frequent mutation and activation of TK genes and signaling pathways. While ABL1 tyrosine kinase inhibitors (TKIs) such as dasatinib and imatinib have been added to chemotherapy regimens for Ph+ B-ALL, over half of these patients will still relapse, which correlates with residual disease burden in the bone marrow (BM) following induction therapy. Hence, new therapeutic strategies are needed for patients with Ikaros-mutant, high-risk Ph+ and Ph– B-ALL.Using mice with a conditional Ikzf1 mutation (Ike5fl) where the recombined allele is similar to the dominant-negative Ik6 mutant found in human B-ALL, we demonstrated recently that Ikaros DNA-binding function is required in the B-lymphoid lineage for transition from the large to small pre-B cell stage of differentiation, and that arrest at this stage of development can give rise to B-ALL (Joshi et al., Nat. Immunol. 2014;15:294). The survival and proliferation of Ikaros mutant pre-B cells is dependent on increased integrin-mediated stromal adhesion and activation of focal adhesion kinase (FAK). FAK is a non-receptor TK, downstream of integrins and growth factor receptors, which plays important roles in cancer stem cell biology, the tumor microenvironment and tumorigenesis. VS-4718 and VS-6063 (defactinib) are potent, orally bioavailable FAK inhibitors that inhibit tumor growth and metastasis in preclinical models, and are currently under evaluation in clinical trials in patients with various solid tumors. VS-6063 has demonstrated tolerability and preliminary signs of clinical activity as a single agent and in combination with paclitaxel in phase I trials (ASCO, 2014). Here, we show that BCR-ABL1 cooperates with Ikzf1 mutation to accelerate B-leukemogenesis in mice. BCR-ABL1+ Ikaros-mutant B-ALLs exhibit stroma-mediated resistance to ABL1 TKIs, while the FAK inhibitors VS-4718 and VS-6063 are effective in blocking stromal adhesion and inducing apoptosis in both mouse and human Ikaros-mutant B-ALL samples.To test whether dysregulation of TK signaling cooperates with Ikzf1 mutation in the pathogenesis of high-risk B-ALL, we isolated BM B-lymphoid progenitor cells from wild-type (WT), IkE5fl/+CD2-Cre, and IkE5fl/flCD2-Cre donors, transduced them with BCR-ABL1 retrovirus and transplanted the cells into recipient mice. We observed a dramatic acceleration of precursor B-lymphoid leukemia induced by BCR-ABL1 in IkE5Δ/+ and particularly in IkE5Δ/Δ donor cells that correlated with a striking (~30-fold) increase in the frequency of engrafting leukemia-initiating or leukemic stem cells (LSCs). Relative to Ikzf1 WT BCR-ABL1+ leukemic cells, Ikzf1-mutant BCR-ABL1+ blasts showed significant resistance to imatinib and dasatinib that was dependent on the presence of OP9 stroma.The effect of FAK inhibition, using the FAK inhibitors VS-4718, VS-6062, and VS-6063 (Verastem), was first tested on murine B-ALL cells (genotypes Ikzf1 mutant, Ikzf1 mutant BCR-ABL1+, and Ikzf1 WT BCR-ABL1+) grown on OP9 stroma. FAK inhibitor treatment abolished stromal adhesion of Ikzf1-mutant B-ALL and induced apoptosis in non-adherent cells, but had little effect on Ikzf1 WT B-ALL cells. VS-4718 and VS-6063 were each synergistic with dasatinib in reducing the viability of Ikzf1-mutant BCR-ABL1+ B-ALL cells cultured on OP9 stroma. For primary human B-ALL samples grown on OP9 stroma, IKZF1-mutant cells were also more sensitive to FAK inhibitor treatment than WT IKZF1 WT B-ALL, with or without BCR-ABL1 expression.Collectively, these observations suggest a new model to explain the pathogenesis of high-risk B-ALL and its resistance to therapy. B-ALLs with IKZF1 mutations may be resistant to TKIs and to chemotherapy by virtue of their stromal adhesion phenotype, resulting in failure to eliminate BM LSCs. Inhibition of FAK signaling in Ph+ or Ph­–IKZF1-mutant B-ALL may reverse the stromal-mediated resistance to ABL1 TKIs and/or chemotherapy. Therefore, FAK inhibitors warrant further investigation for the treatment of high-risk IKZF1-mutant B-ALL patients. DisclosuresJoshi:Verastem: Research Funding. Yoshida:Verastem, Inc.: Research Funding. Paraskevopoulou:Verastem, Inc.: Research Funding. Zhang:Verastem, Inc.: Research Funding. Krause:Glycomimetics. Inc.: Research Funding. Shapiro:Verastem: Employment, Equity Ownership. Weaver:Verastem: Employment, Equity Ownership. Pachter:Verastem Inc.: Employment, Equity Ownership. Georgopoulos:Verastem, Inc.: Research Funding.

Preclinical Studies of the IAP Antagonist Debio 1143 in Combination with Cytarabine or Doxorubicin in a Mouse Model of AML

Background: Relapse and drug resistance remains the major cause of therapy failure in Acute Myeloid Leukemia (AML). Inhibitors of apoptosis proteins (IAPs) are a family of proteins that suppress proapoptotic signaling and represent attractive targets to overcome chemotherapy resistance. Debio 1143 (a.k.a. AT-406) is an orally-active IAP antagonist able to promote apoptosis alone and/or in combination with chemotherapy. We recently reported the results of a Phase 1 study of Debio 1143 in combination with daunorubicin and cytarabine (araC) in poor-risk AML patients (DiPersio et al. J Clin Oncol 32:5s, 2014, a7029). Although Debio 1143 showed good tolerability and could be safely combined with chemotherapy, 62% (8/13) of patients with complete remissions relapsed within the study period. Here, we used a preclinical AML model to begin examining the effects of BM stroma on the efficacy of Debio 1143 combined with araC or doxorubicin (dox).Methods: We generated murine acute promyelocytic leukemia (APL) cell lines that are resistant to araC and dox both in vitro and in vivo by chronically exposing APL cells to increasing concentrations of drug in vitro. This APL model was previously generated by knocking in the human PML-RARa cDNA into the cathepsin G locus (Westervelt et al. Blood, 2003). We used WST-1 cell proliferation assays and bioluminescence imaging (BLI) to evaluate the efficacy of Debio 1143 to induce apoptosis alone and in combination with araC or dox in vitro in chemotherapy sensitive and resistant APL cells in the presence and absence of M2-10B4 stromal cells. Dox or araC was provided for (a) a 6h period initiated 24h before Debio 1143 (i.e., with an intervening 18h washout of the chemotherapy), (b) simultaneously with Debio 1143, or (c) 6h after Debio 1143. To assess Debio 1143 in vivo, we injected APL cells into syngeneic mice. In our first experiment, mice (n=5/cohort) were treated with PBS, araC (500 mg/kg, s.c.), dox (50 mg/kg, s.c.), Debio 1143 (100 mg/kg, p.o.) or their combination on days 5, 6, 12, and 13 after APL injection. In our second experiment, mice (n=8/cohort) were treated with PBS, araC (200 mg/kg, s.c.), dox (10 mg/kg, s.c.), Debio 1143 (100 mg/kg, p.o.) or their combination on days 3 to 7 and 10 to 16 after APL injection. In both experiments, Debio 1143 was given 2 hours prior to chemotherapy.Results: Figure 1 shows that coculture of APL cells with stromal cells protects them from araC-induced killing (Figure 1A) and that our araC-resistant cells are indeed resistant to araC (Figure 1B). Treatment with Debio 1143 alone has no effect on parental or araC-resistant APL survival in the presence or absence of stroma cells. However, Debio 1143 in combination with araC killed the parental APL cells and overcame the stroma cell-mediated chemotherapy protection (Figure 1A). Although combination therapy partially inhibited araC-resistant APL cell proliferation in the absence of stroma, it had no effect when the cells were cultured in the presence of stroma (Figure 1B). We obtained similar results with dox and dox-resistant cells (data not shown). This in vitro data with the parental APL cell line was confirmed in vivo. Combination therapy with chemotherapy (araC or dox) and Debio 1143 for 4 days (EXP#1) or 10 days (EXP#2) significantly prolonged the overall survival of mice by 4 to 7 days compared to single agent therapy (Debio 1143, araC, or dox) in this aggressive tumor model (Table 1). Preliminary in vitro data suggests that pretreatment of parental or araC-resistant APL cells with chemotherapy (araC or dox) enhances their killing by Debio 1143 compared to simultaneous treatment.Conclusions: Debio 1143 combined with chemotherapy inhibits parental and chemotherapy-resistant APL growth in vitro and in vivo. Future mechanistic studies are required to determine how Debio 1143 can overcome stroma-mediated resistance. [Display omitted] [Display omitted] DisclosuresRettig:Debiopharm International SA: Research Funding. Vuagniaux:Debiopharm International SA: Employment. Zanna:Debiopharm International SA: Employment. DiPersio:Debiopharm International SA: Research Funding.

Exosome Transfer from Stromal to Breast Cancer Cells Regulates Therapy Resistance Pathways

Stromal communication with cancer cells can influence treatment response. We show that stromal and breast cancer (BrCa) cells utilize paracrine and juxtacrine signaling to drive chemotherapy and radiation resistance. Upon heterotypic interaction, exosomes are transferred from stromal to BrCa cells. RNA within exosomes, which are largely noncoding transcripts and transposable elements, stimulates the pattern recognition receptor RIG-I to activate STAT1-dependent antiviral signaling. In parallel, stromal cells also activate NOTCH3 on BrCa cells. The paracrine antiviral and juxtacrine NOTCH3 pathways converge as STAT1 facilitates transcriptional responses to NOTCH3 and expands therapy-resistant tumor-initiating cells. Primary human and/or mouse BrCa analysis support the role of antiviral/NOTCH3 pathways in NOTCH signaling and stroma-mediated resistance, which is abrogated by combination therapy with gamma secretase inhibitors. Thus, stromal cells orchestrate an intricate crosstalk with BrCa cells by utilizing exosomes to instigate antiviral signaling. This expands BrCa subpopulations adept at resisting therapy and reinitiating tumor growth.