Genomic Instability and Activation Of The DNA Damage Response Pathway Is An Independent Predictor Of Poor Prognosis, Is Associated With MYC Expression and Is a Promising Target For Therapy In Diffuse Large B Cell Lymphoma

Genomic instability and constitutive activation of the DNA damage response (DDR) pathway has been recently described in models of aggressive myc-driven lymphoid malignancies. The MYC oncogene has been reported to induce genomic instability by a mechanism involving replication stress. On the other hand, MYC is overexpressed in a fraction of diffuse large B-cell lymphomas (DLBCLs), and its overexpression has been reported to be associated with poor prognosis. The checkpoint kinases 1 (CHK1) and 2 (CHK2), are serine-threonine kinase involved in the DDR pathway. DDR activation triggers the phosphorylation of the histone H2AX at ser 139, a known marker of DNA damage and genomic instability. The correlation between genomic instability, MYC expression, and prognosis has not been investigated yet in DLBCL.

Immunohistochemistry (IHC) for phospho (γ) H2AX, pCHK1, pCHK2 was performed in tissue microarrays (TMAs) from 97 consecutive patients treated at our Institution between 2004 and 2011 with R-CHOP/CHOP-like regimens, with available paraffin embedded tissue from initial diagnosis. Moreover, to evaluate the therapeutic potential of DDR pathway inhibition in DLBCL, the DLBCL cell lines HBL-1, U2932, TMD8, SUDHL-6, BJAB, SUDHL-4 and primary DLBCL cells were incubated with the CHK inhibitor PF-0477736 (Pfizer).

In the TMA study 57% of patients (n=55) displayed high levels of basal γH2AX (>30% of positive cells), 55% (n=53) displayed pCHK1/pCHK2 activation and of note all DLBCL cell lines showed detectable baseline activation of CHK1/CHK2 and/or H2AX phosphorylation, by western immunoblotting. γH2AX positive cases distributed equally in germinal center (GC) and in non GC DLBCLs, and were significantly associated with MYC expression (p<0.01). Five-year survival rate was 70% vs 41% for γH2AX-low and γH2AX-high patients respectively (p=0.01). Factors significantly related to the outcome in multivariate analysis were International Prognostic Index (IPI) score and γH2AX expression. Remarkably the prognostic significance of γH2AX was particularly evident in the low risk IPI group (0-2 risk factors), identifying a subgroup characterized by worse outcome (54% 5-year OS). In the in vitro study a significant growth inhibition (WST-1 assay), was evident after 48 hrs in all cell lines (IC50 10-230 nM). PF-0477736 25-500 nM induced cell death by apoptosis (annexin V- propidium iodide staining) in a time and dose dependent manner. Notably PF-0477736 demonstrated activity also in primary DLBCL cells (IC 50 of 50-500 nM, 24 hrs). We observed inhibition of phosphorylation of the downstream target CDC25c (ser 216), coupled with a marked increase in γH2AX ser 139 and CHK1 phosphorylation (ser317 and 345) following treatment.

A significant fraction of DLBCLs shows high levels of inherent genomic instability; the DDR activation marker γH2AX is a poor prognostic predictor in DLBCL and interestingly is significantly associated with MYC expression. DDR inhibition resulted to be highly effective in DLBCL cell lines and primary DLBCL cells; on treatment modifications of CHK1 and H2AX phosphorylation could be useful biomarkers of CHK inhibitors activity. These data provide strong rationale for targeting the DDR pathway and for clinical investigation of CHK inhibitors in DLBCL.

No relevant conflicts of interest to declare.