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Identifying and Overcoming Mechanisms of Histone Deacetylase Inhibitor Resistance

Reduced expression of pro-apoptotic protein Bim was observed in a skin biopsy sample obtained from a patient with CTCL at the time of disease progression on romidepsin.
Reduced expression of pro-apoptotic protein Bim was observed in a skin biopsy sample obtained from a patient with CTCL at the time of disease progression on romidepsin.

Histone deacetylase inhibitors (HDIs), such as romidepsin, can inhibit the growth of cancer cells and induce their apoptosis by increasing histone acetylation and altering gene expression. Romidepsin has even been approved by the Food and Drug Administration for the treatment of two types of non-Hodgkin lymphoma, cutaneous T cell lymphoma (CTCL) and peripheral T cell lymphoma. But, as Susan Bates, M.D., in CCR’s Medical Oncology Branch, knows firsthand from her work on phase I and II clinical trials testing romidepsin, many cancers are initially resistant or develop resistance to HDIs. Bates, along with Arup Chakraborty, Ph.D., a postdoctoral fellow in her lab, and their colleagues are interested in understanding cellular mechanisms of HDI resistance with the hope of identifying additional pathways that could be targeted to enhance the anticancer efficacy of HDIs.

The researchers began their studies by generating romidepsin-resistant cell lines. While romidepsin is a target of the drug transporter P-glycoprotein (Pgp), upregulation has not been observed in clinical samples obtained from patients with CTCL. To focus on other mechanisms, the scientists selected three resistant HuT78 CTCL sublines, DpVp35, DpVp50, and DpP75, using an approach that prevented Pgp overexpression. These sublines were more resistant to 48-hour treatment with romidepsin and to other HDIs than parental HuT78 cells.

Using a custom-made array, the investigators looked for changes in gene expression between the parental and resistant cells to identify potential resistance mechanisms. They found significantly increased expression of the insulin receptor in the resistant cells. Downstream of the insulin receptor, activated MEK levels were at least four fold higher in the resistant cells compared to parental cells, with decreased STAT3 and Akt phosphorylation. These results suggested that increased MEK activity may mediate resistance to romidepsin.

To test this idea, the researchers incubated the parental and resistant cells with three different MEK inhibitors (MEKi). Few of the parental cells underwent apoptosis, even when exposed to high concentrations of MEKi. The resistant cells, however, were particularly sensitive to MEK inhibition with high levels of annexin V staining and cleaved PARP observed at the lowest MEKi concentrations. In contrast, the resistant cells were insensitive to PI3K and insulin receptor inhibitors. With a combination of romidepsin and 1 or 3nM of a MEKi, significantly more of the cells underwent apoptosis than with the MEKi alone, and the studies suggested that the combination can overcome romidepsin resistance.

The researchers then looked for targets downstream of MEK that might mediate its protective effect. Expression of the proapoptotic protein Bim is known to be negatively regulated by the MAPK pathway, and the investigators found reduced Bim levels in the resistant cells compared to parental cells.

To determine whether Bim expression correlated with apoptosis, the scientists treated resistant cells with MEKi and measured Bim and cleaved PARP levels. In the presence of the inhibitor, Bim protein was restored to the level found in parental cells, and the researchers observed a corresponding increase in PARP cleavage. The investigators then knocked down Bim expression in resistant cells using a small hairpin RNA (shRNA). These cells showed reduced Bim and cleaved PARP levels after MEKi treatment, and more cells survived in the presence of MEKi. Likewise, parental cells transfected with the shRNA had reduced Bim expression and increased resistance to romidepsin treatment, suggesting that Bim is critical for MEKi-induced apoptosis in resistant cells and romidepsin-induced apoptosis in parental cells.

To assess whether similar pathways are implicated in patient responses to HDIs, the investigators examined samples from patients with CTCL participating in a clinical trial of romidepsin. They examined RNA isolated from peripheral blood mononuclear cells obtained from patients before or after romidepsin treatment. After exposure to the HDI, some MAPK-regulated genes were induced while others were suppressed, supporting the idea that alteration of the MAPK pathway may affect cancer cell sensitivity to romidepsin. In a pilot analysis, higher Bim expression levels in patient skin biopsies before romidepsin treatment appeared to correlate with increased sensitivity to romidepsin treatment. Taken together these results suggest that the combination of a MEKi and romidepsin is a promising strategy for enhancing this HDI’s efficacy.

Reference
Chakraborty AR, Robey RW, Luchenko VL, Zhan Z, Riekarz RL, Gillet JP, Kossenkov AV, Wilkerson J, Showe LC, Gottesman MM, Collie NL, Bates SE. MAPK Pathway Activation Leads to Bim Loss and Histone Deacetylase Inhibitor Resistance: Rationale to Combine Romidepsin with a MEK Inhibitor. Blood. March 26, 2013. Pubmed Link