Our present studies indicate that treatment of cancer cells with clinically-relevant, low doses of DAC and AZA can exert sustained changes in gene expression, and critical signaling pathways involved in tumorigenesis, without inducing immediate cytotoxic effects such as DNA damage, apoptosis, and cell cycle arrest. Our preclinical use of transient, 3 day exposure to such doses ex vivo
produces a “memory” type of anti-tumor response in mice bearing transplanted tumor xenografts, which may resemble the prolonged time to response seen in patients with hematologic neoplasms (Kantarjian et al., 2006
; Oki et al., 2008
; Silverman et al., 2002
). In patient tumors and mammospheres, the anti-tumor response occurred more quickly and appeared to be more sensitive to AZA than that observed for cancer cell line generated tumors. This may be explained by the fact that the human tumors were exposed to a longer, more sustained treatment and that primary tissues may be initially more sensitive to AZA. All of these above dynamics following transient drug exposure are accompanied by genome-wide, prolonged gene promoter DNA de-methylation and sustained increases in gene expression, some which occur for key tumor suppressor genes in leukemia and breast cancer cells.
There are several reasons to suggest that at least one key mechanism underlying the anti-tumor responses we demonstrate may involve a resetting of abnormal epigenetic states in treated cancer cells. First, we have achieved a sustained change in the pattern of gene expression without changing primary DNA sequence. Second, as defined for an epigenetic change, these sustained changes persist for significant periods of time after a transient, subsequently withdrawn, signal - ie, in this case, drug treatment. Third, the new expression patterns are accompanied by a new cellular phenotype, anti-tumor effects. Importantly, we probably have brought out these aspects of DAC and AZA treatment by using doses that do not acutely kill cells and, thus, allow the sustained alterations in both gene expression patterns and appearance of a new phenotype to emerge. Importantly, these changes include anti-tumor events in multiple key pathways, such as apoptosis, increased lineage commitment, down-regulation of cell cycling, and others, which continue well after drug removal. Reprogramming might, then, be considered a very desirable type of targeted therapy that can blunt multiple tumor signaling pathways simultaneously.
Perhaps, one of the most striking effects observed in our study concerns the fact that nanomolar doses of both DAC and AZA appear to target, in both cell lines and primary samples of leukemia and breast cancers, self-renewing and/or tumorigenic cell subpopulations. This involves stem-like CD34+
cells in leukemia and CD44+
and mammosphere forming cells in breast cancer. These findings should be considered in the context that one of the most important problems in cancer therapy is the failure of most therapies to target such subpopulations that are most responsible for sustained tumor cell renewal (Jordan et al., 2006
). Our findings suggest perhaps that the often prolonged time course to response in patients with myelodysplasia or frank AML might involve a progressive exhaustion of such cell populations. Such a hypothesis is supported by recent reports that depleting DNMT1, by non-pharmacologic means, in normal mouse hematopoietic and human epithelial cells blocks self-renewal and proper cell maturation leading to cellular depletion of progenitor cells (Broske et al., 2009
; Sen et al., 2010
; Trowbridge et al., 2009
). DAC and AZA do deplete DNMT1 in leukemia and breast cancer cells for variable time periods after transient exposure (). However, the mechanisms underlying our pharmacologically induced responses are certainly more complex since both DAC and AZA inhibit the catalytic sites of DNMT3a and 3b as well. Also, all the DNMT’s assuredly participate in complex protein interactions where they may exert scaffolding functions with effects on other chromatin regulatory features (Robertson et al., 2000
; Rountree et al., 2000
). They also, experimentally, have effects for repressing gene expression which may not require their directly catalyzing DNA methylation (Bachman et al., 2001
). This complexity may actually give DAC and AZA an important advantage in treating cancer cells where they may target all of these functions and their role in governing the epigenetic aberrancies present in cancer. If so, the activities we find for low nanomolar doses, may be very important in rendering these drugs as “gold standards” for the development of agents to target DNA methylation, and other epigenetic abnormalities, as an anti-cancer strategy to inhibit the most tumorigenic subpopulations of cells.
In summary, our findings have much relevance for strategies to use DAC and AZA more widely for the management of cancer. These drugs are already making an impact for patients with hematological malignancies. Our findings now suggest ways and biomarkers that might be used to predict and/or monitor clinical efficacy. The similar “memory” type of responses we find for primary and cultured breast cancer cells, and the key pathway changes seen, indicate that the treatment of many cancers may be considered and with doses that will be not only efficacious but also minimally toxic to patients. Such possibilities are emerging in our recently completed Lung Cancer SPORE and Stand-up to Cancer (SU2C) sponsored trial for patients with advanced non-small cell lung carcinoma (Juergens et al.). SU2C trials have now begun in breast cancer which might be well informed by the pre-clinical studies we now report. Moreover, it is especially appealing to consider that DAC and AZA might sensitize tumor cells to other drugs, as is suggested in Juergens et al., that target the oncogenic pathways we have shown to be altered and allow use of reduced, less toxic, doses for these other agents.