Abstract Overexpression of the “activating” transcription factors, E2F1,2 and-3a induces genes involved in DNA synthesis and leads to cellular proliferation, tumor growth, and invasion. Therefore, inhibiting the overexpression of one or more activating E2Fs is a recognized target in cancer therapeutics. Dysregulation/overexpression of E2Fs is also controlled by deletion or mutations in the retinoblastoma protein in tumors. In our previous studies we showed that a novel penetratin conjugated 7-mer peptide (PEP) inhibited transcription of the E2F1, 2 and 3a by bounding tightly to E2F promoters. The PEP was cytotoxic at low micro molar concentrations to several malignant cell lines. As the PEP was unstable in vivo, the PEP was encapsulated in PEGylated liposomes (PL-PEP). Treatment of xenografts of the pRB negative small cell lung cancer H-69 and castrate resistant prostate cancer DU145 tumors propagated in mice with the PL- PEP, caused tumor regression. To increase stability and potency of the PEP, L-Arg in the PEP was replaced by D-Arg. Molecular simulation studies showed that the D-Arg PEP secondary structure is more stable than the L- Arg peptide structure in water. In vitro studies showed that the D-Arg PEP was more potent and more resistant to degradation by serum proteases than the L- form. As E2F is important for DNA repair, and for transcription of thymidylate synthase, we tested the effects of the PEP in combination with cisplatin, a DNA damaging drug and pemetrexed, a potent thymidylate synthase inhibitor. Combination studies of the D-ARG pep with cisplatin (in DU145, PC3, LnCap and 4T1) cells and with pemetrexed (in non-small cell lung cancer, H2009, H441, H1975 and H2228) resulted in synergistic cytotoxicity as determined by Chou-Talalay analysis. Citation Format: Gulam M. Rather, Michael Anyanwu, John E. Kerrigan, Kathleen W. Scotto, Olga Garbuzenko, Tamara Minko, Zoltan Szekely, Joseph R. Bertino. A modified L-penetratin peptide targeting e2f-1, 2 and 3 is effective in combination with cisplatin or pemetrexed against prostate and lung cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5213.