The synthesis of a series of single entity, bifunctional MEK1/PI3K inhibitors achieved by covalent linking of structural analogs of the ATP-competitive PI3K inhibitor ZSTK474 and the ATP-noncompetitive MEK inhibitor PD0325901 is described. Inhibitors displayed potent in vitro inhibition of MEK1 (0.015 < IC50 (nM) < 56.7) and PI3K (54 < IC50 (nM) < 341) in enzymatic inhibition assays. Concurrent MEK1 and PI3K inhibition was demonstrated with inhibitors 9 and 14 in two tumor cell lines (A549, D54). Inhibitors produced dose-dependent decreased cell viability similar to the combined administration of equivalent doses of ZSTK474 and PD0325901. In vivo efficacy of 14 following oral administration was demonstrated in D54 glioma and A549 lung tumor bearing mice. Compound 14 showed a 95% and 67% inhibition of tumor ERK1/2 and Akt phosphorylation, respectively, at 2 h postadministration by Western blot analysis, confirming the bioavailability and efficacy of this bifunctional inhibitor strategy toward combined MEK1/PI3K inhibition.

Endocrine therapy (ET) is an effective first-line therapy for women with estrogen receptor-positive (ER + ) breast cancers. While both ionizing radiation (RT) and ET are used for the treatment of women with ER+ breast cancer, the most effective sequencing of therapy and the effect of ET on tumor radiosensitization remains unclear. Here we sought to understand the effects of inhibiting estrogen receptor (ER) signaling in combination with RT in multiple preclinical ER+ breast cancer models. Clonogenic survival assays were performed using variable pre- and post-treatment conditions to assess radiosensitization with estradiol, estrogen deprivation, tamoxifen, fulvestrant, or AZD9496 in ER+ breast cancer cell lines. Estrogen stimulation was radioprotective (radiation enhancement ratios [rER]: 0.51–0.82). Conversely, when given one hour prior to RT, ER inhibition or estrogen depletion radiosensitized ER+ MCF-7 and T47D cells (tamoxifen rER: 1.50–1.60, fulvestrant rER: 1.76–2.81, AZD9496 rER: 1.33–1.48, estrogen depletion rER: 1.47–1.51). Combination treatment resulted in an increase in double-strand DNA (dsDNA) breaks as a result of inhibition of non-homologous end joining-mediated dsDNA break repair with no effect on homologous recombination. Treatment with tamoxifen or fulvestrant in combination with RT also increased the number of senescent cells but did not affect apoptosis or cell cycle distribution. Using an MCF-7 xenograft model, concurrent treatment with tamoxifen and RT was synergistic and resulted in a significant decrease in tumor volume and a delay in time to tumor doubling without significant toxicity. These findings provide preclinical evidence that concurrent treatment with ET and RT may be an effective radiosensitization strategy.

Standard radiation (RT) therapy does not reliably provide locoregional control for women with multi-node positive and triple-negative (TNBC) breast cancers. We hypothesized that CDK4/6 inhibition (CDK4/6i) would increase the radiosensitivity not only of estrogen receptor positive (ER+) cells, but also TNBC that express retinoblastoma (RB) protein. We found that CDK4/6i radiosensitized RB wild-type TNBC (n=4, rER 1.49 -2.22), but failed to radiosensitize RB-null TNBC (n=3, rER: 0.84 -1.00). RB expression predicted response to CDK4/6i + RT (R 2 =0.84), and radiosensitization was lost in ER+/TNBC cells (rER: 0.88 -1.13) after RB1 knockdown in isogenic and non-isogenic models. CDK4/6i suppressed homologous recombination (HR) in RB wild-type cells, but not in RB-null cells or isogenic models of RB1 loss; HR competency was rescued with RB re-expression. Radiosensitization was independent of non-homologous end joining and the known effects of CDK4/6i on cell cycle arrest. Mechanistically, RB and RAD51 interact in vitro to promote HR repair. CDK4/6i produced RB-dependent radiosensitization in TNBC xenografts, but not in isogenic RB1-null xenografts. Our data provide the preclinical rationale for a clinical trial expanding the use of CDK4/6i + RT to difficult to control RBintact breast cancers (including TNBC) and nominate RB status as a predictive biomarker of therapeutic efficacy.

The structure-based design of a new single entity, MEK/PI3K bifunctional inhibitor (, ), which displays improved MEK1 and PI3K isoform inhibition, is described. demonstrated a 2.2-fold improvement in MEK1 inhibition and a 2.8-, 2.7-, 23-, and 2.5-fold improved inhibition toward the PI3Kα, PI3Kβ, PI3Kδ, and PI3Kγ isoforms, respectively, as compared to a previous lead compound (; ) in enzymatic inhibition assays. demonstrated superior tumoricidal efficacy over in an A375 melanoma spheroid tumor model. was comparatively more effective than in promoting tumor control when administrated orally in a tumor therapy study conducted in an A375 melanoma mouse model confirming its bioavailability and efficacy toward combined MEK1/PI3K inhibition.