Peristromal niches protect lung cancers from targeted therapies through a combined effect of multiple molecular mediators.

Targeted therapies directed against oncogenic signaling addictions, such as inhibitors of ALK in ALK+ NSCLC often induce strong and durable clinical responses. However, they are not curative in metastatic cancers, as some tumor cells persist through therapy, eventually developing resistance. Therapy sensitivity can reflect not only cell-intrinsic mechanisms but also inputs from stromal microenvironment. Yet, the contribution of tumor stroma to therapeutic responses in vivo remains poorly defined. To address this gap of knowledge, we assessed the contribution of stroma-mediated resistance to therapeutic responses to the frontline ALK inhibitor alectinib in xenograft models of ALK+ NSCLC. We found that stroma-proximal tumor cells are partially protected against cytostatic effects of alectinib. This effect is observed not only in remission, but also during relapse, indicating the strong contribution of stroma-mediated resistance to both persistence and resistance. This therapy-protective effect of the stromal niche reflects a combined action of multiple mechanisms, including growth factors and extracellular matrix components. Consequently, despite improving alectinib responses, suppression of any individual resistance mechanism was insufficient to fully overcome the protective effect of stroma. Focusing on shared collateral sensitivity of persisters offered a superior therapeutic benefit, especially when using an antibody-drug conjugate with bystander effect to limit therapeutic escape. These findings indicate that stroma-mediated resistance might be the major contributor to both residual and progressing disease and highlight the limitation of focusing on suppressing a single resistance mechanism at a time.

Stroma-Mediated Breast Cancer Cell Proliferation Indirectly Drives Chemoresistance by Accelerating Tumor Recovery between Chemotherapy Cycles.

The ability of tumors to survive therapy reflects both cell-intrinsic and microenvironmental mechanisms. Across many cancers, including triple-negative breast cancer (TNBC), a high stroma/tumor ratio correlates with poor survival. In many contexts, this correlation can be explained by the direct reduction of therapy sensitivity induced by stroma-produced paracrine factors. We sought to explore whether this direct effect contributes to the link between stroma and poor responses to chemotherapies. In vitro studies with panels of TNBC cell line models and stromal isolates failed to detect a direct modulation of chemoresistance. At the same time, consistent with prior studies, fibroblast-produced secreted factors stimulated treatment-independent enhancement of tumor cell proliferation. Spatial analyses indicated that proximity to stroma is often associated with enhanced tumor cell proliferation in vivo. These observations suggested an indirect link between stroma and chemoresistance, where stroma-augmented proliferation potentiates the recovery of residual tumors between chemotherapy cycles. To evaluate this hypothesis, a spatial agent-based model of stroma impact on proliferation/death dynamics was developed that was quantitatively parameterized using inferences from histologic analyses and experimental studies. The model demonstrated that the observed enhancement of tumor cell proliferation within stroma-proximal niches could enable tumors to avoid elimination over multiple chemotherapy cycles. Therefore, this study supports the existence of an indirect mechanism of environment-mediated chemoresistance that might contribute to the negative correlation between stromal content and poor therapy outcomes. SIGNIFICANCE: Integration of experimental research with mathematical modeling reveals an indirect microenvironmental chemoresistance mechanism by which stromal cells stimulate breast cancer cell proliferation and highlights the importance of consideration of proliferation/death dynamics. See related commentary by Wall and Echeverria, p. 3667.

Paracrine enhancement of tumor cell proliferation provides indirect stroma-mediated chemoresistance via acceleration of tumor recovery between chemotherapy cycles.

The ability of tumors to survive therapy reflects both cell-intrinsic and microenvironmental mechanisms. Across many cancers, including triple-negative breast cancer (TNBC), a high stroma/tumor ratio correlates with poor survival. In many contexts, this correlation can be explained by the direct reduction of therapy sensitivity by stroma-produced paracrine factors. We sought to explore whether this direct effect contributes to the link between stroma and poor responses to chemotherapies. Our in vitro studies with panels of TNBC cell line models and stromal isolates failed to detect a direct modulation of chemoresistance. At the same time, consistent with prior studies, we observed treatment-independent enhancement of tumor cell proliferation by fibroblast-produced secreted factors. Using spatial statistics analyses, we found that proximity to stroma is often associated with enhanced tumor cell proliferation in vivo . Based on these observations, we hypothesized an indirect link between stroma and chemoresistance, where stroma-augmented proliferation potentiates the recovery of residual tumors between chemotherapy cycles. To evaluate the feasibility of this hypothesis, we developed a spatial agent-based model of stroma impact on proliferation/death dynamics. The model was quantitatively parameterized using inferences from histological analyses and experimental studies. We found that the observed enhancement of tumor cell proliferation within stroma-proximal niches can enable tumors to avoid elimination over multiple chemotherapy cycles. Therefore, our study supports the existence of a novel, indirect mechanism of environment-mediated chemoresistance that might contribute to the negative correlation between stromal content and poor therapy outcomes.

Spontaneous cell fusions as a mechanism of parasexual recombination in tumour cell populations.

The initiation and progression of cancers reflect the underlying process of somatic evolution, in which the diversification of heritable phenotypes provides a substrate for natural selection, resulting in the outgrowth of the most fit subpopulations. Although somatic evolution can tap into multiple sources of diversification, it is assumed to lack access to (para)sexual recombination-a key diversification mechanism throughout all strata of life. On the basis of observations of spontaneous fusions involving cancer cells, the reported genetic instability of polypoid cells and the precedence of fusion-mediated parasexual recombination in fungi, we asked whether cell fusions between genetically distinct cancer cells could produce parasexual recombination. Using differentially labelled tumour cells, we found evidence of low-frequency, spontaneous cell fusions between carcinoma cells in multiple cell line models of breast cancer both in vitro and in vivo. While some hybrids remained polyploid, many displayed partial ploidy reduction, generating diverse progeny with heterogeneous inheritance of parental alleles, indicative of partial recombination. Hybrid cells also displayed elevated levels of phenotypic plasticity, which may further amplify the impact of cell fusions on the diversification of phenotypic traits. Using mathematical modelling, we demonstrated that the observed rates of spontaneous somatic cell fusions may enable populations of tumour cells to amplify clonal heterogeneity, thus facilitating the exploration of larger areas of the adaptive landscape (relative to strictly asexual populations), which may substantially accelerate a tumour's ability to adapt to new selective pressures.

Resistance to targeted therapies as a multifactorial, gradual adaptation to inhibitor specific selective pressures.

Despite high initial efficacy, targeted therapies eventually fail in advanced cancers, as tumors develop resistance and relapse. In contrast to the substantial body of research on the molecular mechanisms of resistance, understanding of how resistance evolves remains limited. Using an experimental model of ALK positive NSCLC, we explored the evolution of resistance to different clinical ALK inhibitors. We found that resistance can originate from heterogeneous, weakly resistant subpopulations with variable sensitivity to different ALK inhibitors. Instead of the commonly assumed stochastic single hit (epi) mutational transition, or drug-induced reprogramming, we found evidence for a hybrid scenario involving the gradual, multifactorial adaptation to the inhibitors through acquisition of multiple cooperating genetic and epigenetic adaptive changes. Additionally, we found that during this adaptation tumor cells might present unique, temporally restricted collateral sensitivities, absent in therapy naïve or fully resistant cells, suggesting the potential for new therapeutic interventions, directed against evolving resistance.

Novel role of pleckstrin homology domain of the Bcr-Abl protein: analysis of protein-protein and protein-lipid interactions.

The Bcr-Abl protein is a marker for malignant transformation in chronic myeloid leukemia and in acute lymphoblastic leukemia. There are three Bcr-Abl chimeras known so far, p190, p210 and p230. The only structural difference between the three Bcr-Abl proteins is the presence of DH and PH domains from the Bcr gene in p210 and p230. The Bcr-Abl DH domain is functioning as a guanine nucleotide exchange factor for Rho family of small GTPases. The PH domain confers binding to phosphoinositides but some PH domains have also been found to bind specific target proteins. Here we show that the PH domain from Bcr-Abl binds a number of proteins involved in vital cellular processes. These proteins include PLCvarepsilon, Zizimin1, tubulin and SMC1. The revelation of the role of the Bcr-Abl PH domain in leukemogenesis is likely to provide clues to the molecular mechanisms underlying the phenotypes of Bcr-Abl positive leukemia and could therefore provide tools for the identification of targets for the development of therapeutic treatments.