Abstract #5550: MLH1 has a combinatorial role with MGMT in determining resistance to chemotherapy in DLBCL
Authors: Violetta Leshchenko, Pei-Yu Kuo, Ari Melnick, Samir Parekh
Journal: Cancer Research (2009)

AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO

Temozolomide (TMZ) is an orally administered alkylating agent for the treatment of malignant gliomas. Diffuse Large B Cell Lymphoma (DLBCL) is the most common subtype of Non- Hodgkin\#8217;s Lymphoma. Since TMZ was shown to have activity in Primary CNS lymphoma, and DLBCL is responsive to alkylating agents, we hypothesized that TMZ could have activity in DLBCLs. It is important to note that response to TMZ is associated with epigenetic silencing of Methyl-guanine DNA methyltransferase (MGMT), a DNA repair protein. We therefore hypothesized that MGMT expression or promoter methylation may predict response in DLBCL as well. We therefore exposed a panel of 15 DLBCL cell lines to different doses of TMZ in both single and multi-day schedules. We divided our panel into two groups \#8220;sensitive\#8221; and \#8220;resistant\#8221; using a single dose IC50 cutoff of 150 µM, which represents the clinically achievable concentration of drug. Using a multi-day dosing schedule consistent with typical clinical regimens, we found cell kill 80%-90% even in the \#8220;TMZ resistant\#8221; cell lines after 5 consecutive daily IC25 doses. To determine whether MGMT expression or promoter methylation could predict resistance or sensitivity amongst the DLBCL cells, we first compared MGMT RNA and protein levels to response to TMZ in these cell lines. All \#8220;TMZ sensitive\#8221; group cell lines expressed some MGMT RNA/protein, however lack of MGMT did not confer sensitivity to TMZ, as 50% of \#8220;TMZ resistant\#8221; cell lines were MGMT negative. Using the MassArray technique, we found that MGMT promoter hypermethylation correlated with MGMT silencing in DLBCL cell lines, but there was no direct correlation of percent promoter methylation to TMZ IC50 values. Since an intact mismatch repair function is critically required to rescue cells from DNA damage from alkylating agents, we further investigated the role of MLH1 (human mutL homolog 1) in determining resistance of DLBCL cell lines to TMZ. We found that \#8220;TMZ sensitive\#8221; group of cell lines had decreased MLH1 protein expression. All MGMT negative cell lines in our panel were found to reciprocally show robust MLH1 protein expression. We therefore hypothesized that MGMT and MLH1 may have combinatorial effects in determining resistance to TMZ in DLBCL cell lines. To test this, we treated both MGMT positive and MGMT negative DLBCL cell lines with small interfering RNA (siRNA) targeting MLH1. Upon sequential treatment with TMZ, we found that MLH1 knock-down increases cell kill in DLBCL cells by TMZ as compared to controls. This effect was more pronounced in MGMT negative DLBCL cell lines, all of which are in the \#8220;TMZ resistant\#8221;. We therefore conclude that MLH1 has a combinatorial role with MGMT in determining resistance to TMZ in DLBCLs. We expect the cell line and murine experiments to provide evidence to support a clinical trial of TMZ in DLBCL patients. Moreover, modulation of MLH1 and MGMT may prove to be of therapeutic benefit in combination with TMZ in this disease.

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 5550.