Background: Diffuse large B cell lymphoma (DLBCL) is the most common type of aggressive lymphoma. Approximately 40% of DLBCL patients have refractory disease or disease that will relapse after an initial response to the combinatorial chemo-immunotherapy. Thus, finding new therapeutic approaches is urgently needed for this lymphoma. Experimental evidence showed that high levels of Myc protein is very common in DLBCL and correlated with poor survival prognosis of DLBCL patients, suggesting that Myc is a crucial target for DLBCL therapy. The checkpoint kinases Chk1 and Wee1 are key cell cycle regulators, required during normal S phase to avoid deleterious DNA breakage, and to maintain cancer cell survival under replication stress. Chk1 inhibitors have been recently shown to be effective in Myc overexpressing cells and are strongly synergistic with Wee1 inhibitors in many experimental systems. We herein investigated the effects of Chk1 and Wee1 inhibition in DLBCL cell lines differently expressing Myc protein. Material and Methods: Eleven cell lines derived from germinal center B-cell like (GCB) and activated B cell like (ABC) DLBCL were treated with the Chk1 inhibitor PF-00477736 and the Wee1 inhibitor AZD-1775 alone and in combination. Drug combination was quantified by calculating the Combination Index (CI) (Chou Talalay method). Cell cycle analysis by FACS and detection of apoptosis by caspase-3 activity were performed after such treatments. Myc protein levels and DNA damage markers were evaluated by Western Blot Analysis. Results: Myc protein levels and the degree of endogenous constitutive DNA damage (yH2AX activation and pS317-Chk1) detected in the different DLBCL cell lines did not correlate with the extent of sensitivity to PF-00477736 and to AZD-1775 (IC50s ranging respectively from 68 to 1023 nM and from 114 to 968 nM). The combined treatment is strongly synergistic in all the DLBCL cell lines, with CI values, evaluated at the IC50 doses, ranging from 0.06 to 0.77. The drug combination induced an S phase delay and an accumulation of DNA damage measured by yH2AX activation. Enhanced caspase-3 activity observed after the combined treatment suggested that cell death by apoptosis is also occurring. Interestingly a strong decrease of Myc protein levels not associated with Myc mRNA down-regulation was observed after the dual inhibition of Chk1 and Wee1. Molecular investigation is undergoing to elucidate the mechanisms at the basis of this Myc protein expression modulation. Conclusions: Overall these data suggest that combining Chk1 and Wee1 inhibitors could be a new and effective therapeutic strategy to test in clinical setting in DLBCL.