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BackgroundThe genetic origins of chemotherapy resistance are well established; however, the role of epigenetics in drug resistance is less well understood. To investigate mechanisms of drug resistance, we performed systematic genetic, epigenetic, and transcriptomic analyses of an alkylating agent-sensitive murine lymphoma cell line and a series of resistant lines derived by drug dose escalation.MethodsDose escalation of the alkylating agent mafosfamide was used to create a series of increasingly drug-resistant mouse Burkitt's lymphoma cell lines. Whole genome sequencing, DNA microarrays, reduced representation bisulfite sequencing, and chromatin immunoprecipitation sequencing were used to identify alterations in DNA sequence, mRNA expression, CpG methylation, and H3K27me3 occupancy, respectively, that were associated with increased resistance.ResultsOur data suggest that acquired resistance cannot be explained by genetic alterations. Based on integration of transcriptional profiles with transcription factor binding data, we hypothesize that resistance is driven by epigenetic plasticity. We observed that the resistant cells had H3K27me3 and DNA methylation profiles distinct from those of the parental lines. Moreover, we observed DNA methylation changes in the promoters of genes regulated by E2a and members of the polycomb repressor complex 2 (PRC2) and differentially expressed genes were enriched for targets of E2a. The integrative analysis considering H3K27me3 further supported a role for PRC2 in mediating resistance. By integrating our results with data from the Immunological Genome Project (Immgen.org), we showed that these transcriptional changes track the B-cell maturation axis.ConclusionsOur data suggest a novel mechanism of drug resistance in which E2a and PRC2 drive changes in the B-cell epigenome; these alterations attenuate alkylating agent treatment-induced apoptosis.

BackgroundThe genetic origins of chemotherapy resistance are well established; however, the role of epigenetics in drug resistance is less well understood. To investigate mechanisms of drug resistance, we performed systematic genetic, epigenetic, and transcriptomic analyses of an alkylating agent-sensitive murine lymphoma cell line and a series of resistant lines derived by drug dose escalation.MethodsDose escalation of the alkylating agent mafosfamide was used to create a series of increasingly drug-resistant mouse Burkitt's lymphoma cell lines. Whole genome sequencing, DNA microarrays, reduced representation bisulfite sequencing, and chromatin immunoprecipitation sequencing were used to identify alterations in DNA sequence, mRNA expression, CpG methylation, and H3K27me3 occupancy, respectively, that were associated with increased resistance.ResultsOur data suggest that acquired resistance cannot be explained by genetic alterations. Based on integration of transcriptional profiles with transcription factor binding data, we hypothesize that resistance is driven by epigenetic plasticity. We observed that the resistant cells had H3K27me3 and DNA methylation profiles distinct from those of the parental lines. Moreover, we observed DNA methylation changes in the promoters of genes regulated by E2a and members of the polycomb repressor complex 2 (PRC2) and differentially expressed genes were enriched for targets of E2a. The integrative analysis considering H3K27me3 further supported a role for PRC2 in mediating resistance. By integrating our results with data from the Immunological Genome Project (Immgen.org), we showed that these transcriptional changes track the B-cell maturation axis.ConclusionsOur data suggest a novel mechanism of drug resistance in which E2a and PRC2 drive changes in the B-cell epigenome; these alterations attenuate alkylating agent treatment-induced apoptosis.

Cyclophosphamide is perhaps the most widely used drug in the treatment of breast cancer. Unfortunately, its use is rarely curative because cyclophosphamide-resistant tumor cell populations rapidly emerge. Our investigations sought to identify at least one mechanism by which cyclophosphamide resistance is effected, thereby providing the rationale for strategies that would reverse or even prevent it. At a minimum, the expectation was that our studies would identify a prognostic determinant that could be used to identify patients for whom cyclophosphamide therapy wound be futile, thus sparing them that therapy and the accompanying untoward effects. Two suspects were identified in preclinical models, viz., the enzymes ALDH3A1 and ALDH1A1. Each was found to catalyze the inactivation of cyclophosphamide. Therefore, the more there was of either of these enzymes in cancer cells, the better able they were to defend themselves against this drug. Amounts of these enzymes in clinical breast cancer samples were found to vary over a 300-fold range. Moreover, ALDH3A1 levels could be increased more than 200-fold in cultured breast cancer cells by exposing them to any of several chemicals that are commonly present in various foods and beverages, e.g., broccoli and coffee. Three agents, each potentially useful in overcoming ALDH3A1-mediated cyclophosphamide-resistance, were identified. Retrospective studies established that ALDH1A1 levels in clinical breast cancers were of sufficient magnitude to affect the clinical outcome of cyclophosphamide-based chemotherapy, whereas those of ALDH3A1 were not.

Cyclophosphamide is widely used to kill breast cancer cells that detach from primary tumors prior to the surgical removal of the latter and that relocate in various parts of the body where they usually cannot be detected and where they multiply. Unfortunately, even though cyclophosphamide is amongst the best available drugs for this purpose, its use rarely results in cures because, amongst these relocated cancer cells, there are, or soon are, some that can defend themselves against cytoxan and therefore are not killed by it. The objective of our studies is to find out how such cells do this and, then, to develop strategies that would eliminate, negate or circumvent the defensive measures used by them to survive. Thus far, our investigations suggest support the following: (1) prognostic determinants that can be used to identify patients for whom cyclophosphamide therapy would prove to be futile include cellular levels of ALDH-1 and, perhaps, ALDH-3, (2) clinical ALDH-3 mediated breast cancer cell resistance to cyclophosphamide may be overcome by the co-administration of gossypol or other drugs that inhibit ALDH-3, and (3) consistent with existing information, but yet to be unequivocally established, it may be prudent to avoid certain dietary constituents when taking Cyclophosphamide because they may decrease the sensitivity of breast cancer cells to it by inducing ALDH-3 in these cells.

Cyclophosphamide, mafosfamide and 4-hydroperoxycyclophosphamide are antineoplastic agents collectively referred to as oxazaphosphorines. Each of these is a prodrug, i.e., per se, without cytotoxic activity. Salient features of the metabolic activation of oxazaphosphorines are presented. Oxazaphosphorines are clinically effective; they play a lead role in the treatment of breast cancer until resistant subpopulations become the dominant population. An understanding of how resistance to these agents is effected would likely to be of value because measures may then become apparent as to how to reverse, and/or prevent, it. It is this understanding which is the overall objective of our first-generation investigations.

Cyclophospharnide is the most widely used drug in the treatment of breast cancer. Unfortunately, emergence of drug- resistant tumor cell populations limits its usefulness. Class-I and class-3 aldehyde dehydrogenases (ALDH-1 and ALDH- 3, respectively) have been shown to catalyze the detoxification of cyclophosphamide and other oxazaphosphorines. Predictably, then, relatively elevated levels of ALDH-I and ALDH-3 have been shown to account for resistance to these agents in several cultured breast and other tumor cell models. It follows that cellular resistance to these agents on the part of clinical breast cancers could be due to overexpression of ALDH-1 and/or ALDH-3. Our finding that elevated levels of ALDH-1 and ALDH-3 are present in some primary and metastatic breast tumor tissues supports this notion. Although nearly identical, tumor cell ALDH-3 appears to be different from normal cell ALDH-3. A tumor-specific ALDH-3 would have diagnostic potential. Tumor cell ALDH-3 was found to be more sensitive to inhibition by each of five chlorpropamide analogs than was normal cell ALDH-3 suggesting that selective sensitization of tumor cells to cyclophosphamide and other oxazaphosphorines may be possible when tumor cell insensitivity to these drugs is due to high levels of ALDH-3. Xenobiotics that are abundantly present in the diet/environment, e.g., methylcholanthrene and catechol, rapidly, coordinately and reversibly induced ALDH-3 and other drug metabolizing enzymes in cultured breast land 9ther cancer cell models, thus, rapidly effecting reversible multienzyme-mediated multidrug resistance/collateral sensitivity to cyclophosphamide and certain other anticancer drugs.