tumor volume was calculated using the equation: volume = (length × width 2) × π/6. Additive and synergistic effects were calculated using the FTV method as previously described (59, 60). Study approval. The protocols used in this study were approved by the IACUC of the University of Michigan. Statistics. Statistical analyses were performed using GraphPad Prism 7.0 (GraphPad Software). A log-rank (Mantel-Cox) test was used for survival curve analyses. One-way ANOVA was performed for BC subtype analysis. A 2-sided Student's t test and 1-way ANOVA with Dunnett's multiple comparisons test were used for in vitro statistical analyses. A 1-way ANOVA with Dunnett's multiple comparisons test and a log-rank (Mantel-Cox) test were used for in vivo analyses. A P value of 0.05 or less was considered statistically significant.

Michmerhuizen et al. Seviteronel Radiosensitizes AR+ TNBC RT, suggesting that seviteronel may have a different mechanism of radiosensitization compared to other AR inhibitors. Enzalutamide and seviteronel treatment also had different effects on AR and AR target genes as measured by immunoblot and qPCR. These results implicate AR as a mediator of radioresistance in AR+ TNBC models and support the use of seviteronel as a radiosensitizing agent in AR+ TNBC.

Sustained locoregional control of disease is a significant issue in patients with inflammatory breast cancer (IBC), with local control rates of 80% or less at 5 years. Given the unsatisfactory outcomes for these patients, there is a clear need for intensification of local therapy, including radiation. Inhibition of the DNA repair protein PARP1 has had little efficacy as a single agent in breast cancer outside of studies restricted to patients with BRCA mutations; however, PARP1 inhibition (PARPi) may lead to the radiosensitization of aggressive tumor types. Thus, this study investigates inhibition of PARP1 as a novel and promising radiosensitization strategy in IBC. In multiple existing IBC models (SUM-149, SUM-190, MDA-IBC-3), PARPi (AZD2281-olaparib and ABT-888-veliparib) had limited single-agent efficacy (IC 50 > 10 mmol/L) in proliferation assays. Despite limited single-agent efficacy, submicromolar concentrations of AZD2281 in combination with RT led to significant radiosensitization (rER 1.12-1.76). This effect was partially dependent on BRCA1 mutational status. Radiosensitization was due, at least in part, to delayed resolution of double strand DNA breaks as measured by multiple assays. Using a SUM-190 xenograft model in vivo, the combination of PARPi and RT significantly delays tumor doubling and tripling times compared with PARPi or RT alone with limited toxicity. This study demonstrates that PARPi improves the effectiveness of radiotherapy in IBC models and provides the preclinical rationale for the opening phase II randomized trial of RT AE PARPi in women with IBC (SWOG 1706, NCT03598257).

Purpose: Cyclin-dependent kinase 4/6 (CDK4/6) inhibitors have improved progression-free survival for metastatic, estrogen receptor–positive (ER+) breast cancers, but their role in the nonmetastatic setting remains unclear. We sought to understand the effects of CDK4/6 inhibition (CDK4/6i) and radiotherapy in multiple preclinical breast cancer models. Experimental Design: Transcriptomic and proteomic analyses were used to identify significantly altered pathways after CDK4/6i. Clonogenic assays were used to quantify the radiotherapy enhancement ratio (rER). DNA damage was quantified using γH2AX staining and the neutral comet assay. DNA repair was assessed using RAD51 foci formation and nonhomologous end joining (NHEJ) reporter assays. Orthotopic xenografts were used to assess the efficacy of combination therapy. Results: Palbociclib significantly radiosensitized multiple ER+ cell lines at low nanomolar, sub IC50 concentrations (rER: 1.21–1.52) and led to a decrease in the surviving fraction of cells at 2 Gy (P < 0.001). Similar results were observed in ribociclib-treated (rER: 1.08–1.68) and abemaciclib-treated (rER: 1.19–2.05) cells. Combination treatment decreased RAD51 foci formation (P < 0.001), leading to a suppression of homologous recombination activity, but did not affect NHEJ efficiency (P > 0.05). Immortalized breast epithelial cells and cells with acquired resistance to CDK4/6i did not demonstrate radiosensitization (rER: 0.94–1.11) or changes in RAD51 foci. In xenograft models, concurrent palbociclib and radiotherapy led to a significant decrease in tumor growth. Conclusions: These studies provide preclinical rationale to test CDK4/6i and radiotherapy in women with locally advanced ER+ breast cancer at high risk for locoregional recurrence.

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.

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The effects of vitamin A and/or vitamin D deficiency were studied in an Arf −/− BcR-ABL acute lymphoblastic leukemia murine model. Vitamin D sufficient mice died earlier (p = 0.003) compared to vitamin D deficient (VDD) mice. Vitamin A deficient (VAD) mice fared worst with more rapid disease progression and decreased survival. Mice deficient for vitamins A and D (VADD) had disease progression similar to VAD mice. Regulatory T cells, previously shown to associate with poor BCR-ABL leukemia control, were present at higher frequencies among CD4 + splenocytes of vitamin A deficient vs. sufficient mice. In vitro studies demonstrated 1,25-dihydroxyvitamin D (1,25(OH) 2 VD 3) increased the number of BCR-ABL ALL cells only when co-cultured with bone marrow stroma. 1,25(OH) 2 VD 3 induced CXCL12 expression in vivo and in vitro in stromal cells and CXCL12 increased stromal migration and the number of BCR-ABL blasts. Vitamin D plus leukemia reprogrammed the marrow increasing production of collagens, potentially trapping ALL blasts. Vitamin A (all trans retinoic acid, ATRA) treated leukemic cells had increased apoptosis, decreased cells in S-phase, and increased cells in G 0 /G 1. ATRA signaled through the retinoid X receptor to decrease BCR-ABL leukemic cell viability. In conclusion, vitamin A and D deficiencies have opposing effects on mouse survival from BCR-ABL ALL. Vitamin D deficiency (VDD) affects an estimated 1 billion people in the world across all ethnicities and age groups 1-3. VDD is an independent risk factor for mortality in the general population 4 and almost 60% of children with malignant diseases have suboptimal vitamin D (VD 3) levels 5. Likewise, the world health organization (WHO) estimated 250 million preschool children are vitamin A deficient (VAD), and this increases the risk of disease and death from severe infections. VAD and VDD are not limited to developing countries. Rather, a recent study found that among 45 people tested in Memphis, TN for vitamin A and vitamin D levels, only two individuals had sufficient levels of both vitamins 6. Vitamin D is a fat-soluble vitamin that not only regulates calcium absorption and bone metabolism, but can also regulate cell proliferation, differentiation and the immune response. The biologically active form of vitamin D, 1,25(OH) 2 VD 3 , binds to the vitamin D receptor (VDR) that heterodimerizes with the retinoid X receptor (RXR). This complex then binds to VDR-RXR response elements in target genes to regulate transcription. VDR is highly expressed in intestine, kidney and bone, but also in normal and neoplastic hematopoietic cells and mesenchymal stem cells in bone marrow 7. 1,25(OH) 2 VD 3 can modify embryonic hematopoietic stem and progenitor cell production 8. 1,25(OH) 2 VD 3 inhibits proliferation of mouse and human myeloid leukemia cells 9 and stimulates myeloid cell differentiation into mature macrophages. Indeed, mice with acute myeloid leukemia (AML) when treated with analogs of 1,25(OH) 2 VD 3 survived longer than the untreated mice 10. Moreover, vitam...

Aim: This study evaluated the impact of CYP3A5 genotype and other patient characteristics on sublingual (SL) tacrolimus exposure and compared the relationship with oral administration. Patients & methods: Tacrolimus concentrations were retrospectively collected for adult lung transplant recipients, who were genotyped for CYP3A5*3, CYP3A4*22, CYP3A7*1C, and POR*28. Regression analyses were performed to determine covariates that impacted the SL and oral tacrolimus concentration/dose ratios. Results: An interaction of CYP3A5 genotype and CYP3A inhibitor increased the SL concentration/dose, while cystic fibrosis decreased the SL concentration/dose. The oral concentration/dose was independently associated with these covariates and was increased by serum creatinine and number of tacrolimus doses. Conclusion: This study suggests personalized dosing strategies for tacrolimus likely need to consider characteristics beyond CYP3A5 genotype.