Drug-tolerant State Research Articles

Abstract SY12-02: Dynamic stemness in melanoma

Abstract Unlike most carcinomas, melanomas can be readily propagated in immunodeficient mice or in tissue culture. Thus, the melanoma research community has been able to study tumor cell properties outside of the human host for decades. When grown in an orthotopic environment such as human skin grafted to mice or in synthetic human skin maintained in vitro, melanoma cells isolated from different stages of tumor progression behave similarly to those in patients. Still, a mechanistic understanding of why melanomas are highly motile and invasive, require few exogenous growth factors for survival and growth, and can hijack fibroblasts, endothelial cells or inflammatory and immune cells to "build" a primitive organ, remains unclear. Genetic analyses, particularly the identifications of activating mutations in the BRAF oncogene, have revealed an Achilles tendon in melanoma because highly specific inhibitors such as PLX4032/RG7204 can significantly shrink tumors. However, such inhibitors are not curative, suggesting that there are subsets of tumor cells that survive therapy despite the presence of BRAF mutations in each individual malignant cell. Apparently, the difficulties in sustained therapy response lie in the considerable heterogeneity within tumors, which in part is due to genetic instability leading to distinct phenotypes and in large part due to epigenetic changes in response to signals from the tumor microenvironment. Starting around 2000, our laboratory followed the lead of the leukemia, brain tumor and breast cancer fields on the existence of cancer stem cells. Indeed we identified a rare population of CD20+ cells that had all of the characteristics of cancer stem cells, including the ability to self-renew and differentiate, and the cells were highly tumorigenic in immunodeficient mice (Fang et al, 2005). Their differentiation patterns not only represented those seen among melanoma cells but we could also drive malignant cells into adipogenic, chondrogenic and osteogenic differentiation pathways by simply changing media conditions. Although such differentiation patterns are rarely seen in patients, they do occur and have puzzled pathologists for decades. Our earlier studies on cancer stem cells in melanoma were greatly supported by work from the Frank group on the ABCB5 marker, an ATP-binding cassette transporter (Frank et al, 2005, Schatton et al, 2008) and on the nerve growth factor receptor by the groups of Sommer (Wong et al 2006) and Weissman (Boiko et al, 2010). However, our own studies were hampered by inconsistent expression of CD20 and a high ‘noise’ in the CD20− cells of relatively high tumorigenicity. We therefore focused on a population representing 1 to 5% of all melanoma cells that did not proliferate or proliferated at rates of 20- to 50-fold slower than the main population (Roesch et al. 2010). These cells were characterized by high expression of JARID1B, a member of the H3K4 demethylase family, which is critical in regulating gene expression and cellular identities and is an epigenetic marker for embryonic stem cells. When released from their microenvironment, JARID1B-positive, nonproliferating melanoma cells gave rise to a rapidly proliferating progeny that reconstituted the parental heterogeneity of JARID1B-positive and -negative cells. Stable knockdown of JARID1B led to an initial acceleration of tumor growth followed by exhaustion as determined by serial xenotransplantation in NSG mice, suggesting that JARID1B plays an essential role in continuous melanoma growth. In contrast to studies by other cancer stem cell groups, we used a combination of serial xenotransplantations plus stable enforcement of a distinct (in this case “non-stem like”) cell phenotype to see how tumors that had been fully established can exhaust over time. Notably, when we performed conventional xenotransplantation with “spontaneous” JARID1B-positive vs. -negative phenotypes, there was no significant difference in tumor initiation capacity between both populations. Single melanoma cells of either population were equally tumorigenic. High tumorigenic growth of melanoma cells independent of marker selection had earlier been demonstrated by Morrison and co-workers (Quintana et al. 2008, 2010). Since JARID1B-negative cells also gave rise to positive cells in vitro and in vivo, we assume that stemness in melanoma does not follow a static but rather a dynamic model. Similar plasticity of the phenotype with a strong dependence on signals from the microenvironment (matrix, fibroblasts, endothelial cells, inflammatory cells or simple changes in oxygen tension) could also be observed for other cancer stem cell markers in melanoma. The Settleman laboratory recently pointed to a new direction of clinical importance: JARID1A, a close homologue of JARID1B, is required for drug resistance in non-small cell lung cancer cells, which suggests that slow-cycling cells survive most conventional and targeted therapies and that this subpopulation requires specific targeting. Our own unpublished experiments strongly indicate that the slow-growing JARID1B+ population is highly resistant to a variety of different therapy protocols. In the future we expect the development of two therapies, one for the minor population with stem cell-like properties, the other for the major population representing the bulk of a tumor.

Tumor heterogeneity and strategies to overcome kinase inhibitor resistance in cancer: lessons from melanoma

Tumor heterogeneity and strategies to overcome kinase inhibitor resistance in cancer: lessons from melanoma

Modeling drug sensitivity and resistance in human tumor-derived cell lines

Abstract Accumulating evidence points to the importance of genomic heterogeneity across human tumors as a critical determinant of the variable clinical response to molecularly targeted cancer therapies. We have used cancer cell lines as a model system for examining the relationship between underlying genotypes and sensitivity to candidate anticancer agents. These studies have revealed the utility of large panels of cancer cell lines to capture the genomic heterogeneity of human cancer and to identify clinically relevant genotype-phenotype relationships. These studies have begun to shed light on mechanisms underlying sensitivity as well as de novo and acquired resistance to a variety of small molecule inhibitors. Using this same platform, we have also identified a mechanism of reversible drug “tolerance” that involves a distinct chromatin state displayed transiently by small subpopulations of cancer cells. This drug-tolerant state can be disrupted by chromatin-modifying agents, potentially yielding a therapeutic opportunity to prevent or delay the development of more stable genetically conferred drug resistance mechanisms.

Abstract IA2-1: Modeling drug sensitivity and resistance in human tumor-derived cell lines

Abstract Agccumulating evidence points to the importance of genomic heterogeneity across human tumors as a critical determinant of the variable clinical response to molecularly targeted cancer therapies. We have used cancer cell lines as a model system for examining the relationship between underlying genotypes and sensitivity to candidate anticancer agents. These studies have revealed the utility of large panels of cancer cell lines to capture the genomic heterogeneity of human cancer and to identify clinically relevant genotype- phenotype relationships. These studies have begun to shed light on mechanisms underlying sensitivity as well as de novo and acquired resistance to a variety of small molecule inhibitors. Using this same platform, we have also identified a mechanism of reversible drug tolerance that involves a distinct chromatin state displayed transiently by small subpopulations of cancer cells. This drug-tolerant state can be disrupted by chromatin-modifying agents, potentially yielding a therapeutic opportunity to prevent or delay the development of more stable genetically conferred drug resistance mechanisms. Citation Information: Clin Cancer Res 2010;16(14 Suppl):IA2-1.

A Chromatin-Mediated Reversible Drug-Tolerant State in Cancer Cell Subpopulations

Accumulating evidence implicates heterogeneity within cancer cell populations in the response to stressful exposures, including drug treatments. While modeling the acute response to various anticancer agents in drug-sensitive human tumor cell lines, we consistently detected a small subpopulation of reversibly "drug-tolerant" cells. These cells demonstrate >100-fold reduced drug sensitivity and maintain viability via engagement of IGF-1 receptor signaling and an altered chromatin state that requires the histone demethylase RBP2/KDM5A/Jarid1A. This drug-tolerant phenotype is transiently acquired and relinquished at low frequency by individual cells within the population, implicating the dynamic regulation of phenotypic heterogeneity in drug tolerance. The drug-tolerant subpopulation can be selectively ablated by treatment with IGF-1 receptor inhibitors or chromatin-modifying agents, potentially yielding a therapeutic opportunity. Together, these findings suggest that cancer cell populations employ a dynamic survival strategy in which individual cells transiently assume a reversibly drug-tolerant state to protect the population from eradication by potentially lethal exposures.