Increased rates of locoregional recurrence are observed in patients with basal-like breast cancer (BC) despite the use of radiation therapy (RT); therefore, approaches that result in ...radiosensitization of basal-like BC are critically needed. Using patients' tumor gene expression data from 4 independent data sets, we correlated gene expression with recurrence to find genes significantly correlated with early recurrence after RT. The highest-ranked gene, TTK, was most highly expressed in basal-like BC across multiple data sets. Inhibition of TTK by both genetic and pharmacologic methods enhanced radiosensitivity in multiple basal-like cell lines. Radiosensitivity was mediated, at least in part, through persistent DNA damage after treatment with TTK inhibition and RT. Inhibition of TTK impaired homologous recombination (HR) and repair efficiency, but not nonhomologous end-joining, and decreased the formation of Rad51 foci. Reintroduction of wild-type TTK rescued both radioresistance and HR repair efficiency after TTK knockdown; however, reintroduction of kinase-dead TTK did not. In vivo, TTK inhibition combined with RT led to a significant decrease in tumor growth in both heterotopic and orthotopic, including patient-derived xenograft, BC models. These data support the rationale for clinical development of TTK inhibition as a radiosensitizing strategy for patients with basal-like BC, and efforts toward this end are currently underway.
Increased rates of locoregional recurrence (LR) have been observed in triple negative breast cancer (TNBC) despite multimodality therapy, including radiation (RT). Recent data suggest inhibiting the ...androgen receptor (AR) may be an effective radiosensitizing strategy, and AR is expressed in 15-35% of TNBC tumors. The aim of this study was to determine whether seviteronel (INO-464), a novel CYP17 lyase inhibitor and AR antagonist, is able to radiosensitize AR-positive (AR+) TNBC models. In cell viability assays, seviteronel and enzalutamide exhibited limited effect as a single agent (IC50 > 10 μM). Using clonogenic survival assays, however, AR knockdown and AR inhibition with seviteronel were effective at radiosensitizing cells with radiation enhancement ratios of 1.20-1.89 in models of TNBC with high AR expression. AR-negative (AR-) models, regardless of their estrogen receptor expression, were not radiosensitized with seviteronel treatment at concentrations up to 5 μM. Radiosensitization of AR+ TNBC models was at least partially dependent on impaired dsDNA break repair with significant delays in repair at 6, 16, and 24 h as measured by immunofluorescent staining of γH2AX foci. Similar effects were observed in an
AR+ TNBC xenograft model where there was a significant reduction in tumor volume and a delay to tumor doubling and tripling times in mice treated with seviteronel and radiation. Following combination treatment with seviteronel and radiation, increased binding of AR occurred at DNA damage response genes, including genes involved both in homologous recombination and non-homologous end joining. This trend was not observed with combination treatment of enzalutamide and 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.
Background
: To evaluate the relationship between sleep and next-day physical activity (PA) under free-living conditions in women.
Methods
: Sleep and PA were measured objectively for 7 consecutive ...days by accelerometry in 330 young adult women (aged 17–25 y). A structural equation model was used to evaluate the relationship between the driving factor of sleep (total sleep or morning wake time) and the amount of nonsleep sedentary (SED) and moderate to vigorous physical activity (MVPA) each day.
Results
: With sleep duration as the driving factor, the estimates of β
SED
and β
MVPA
were −0.415 and −0.093, respectively (
P
≤ .05). For every hour slept, a 24.9-minute reduction in SED time and a 5.58-minute reduction in MVPA were observed. With wake time as the driving factor, the estimates of β
SED
and β
MVPA
were −0.636 and −0.149, respectively. For every wake time that was 1 hour later, a 38.2-minute decrease in SED and a 8.9-minute decrease in MVPA (
P
≤ .05) were observed.
Conclusions
: Women who wake later or who sleep longer tend to get less MVPA throughout the day. Getting up earlier and going to bed earlier may support behaviors that improve PA and lifestyle.
Abstract
Background: Radiation therapy (RT) is standard in the treatment of many women with breast cancer (BC). Despite this, women with estrogen receptor positive (ER+) BC respond heterogeneously ...to RT. Radiosensitization methods for aggressive ER+ disease are needed. We performed a radiosensitizer screen paired with transcriptomic and proteomic data from ER+ models treated +/-RT to identify potential mediators of RT resistance.
Methods: Clonogenic survival assays were used to determine RT sensitivity of 21 BCC lines as well as radiosensitization with drug treatment. IC50 values were determined for 130 clinical compounds and correlation coefficients were calculated using IC50 values and SF-2Gy. Microarray and RPPA data was used for differential gene/protein expression and pathway analysis. AlamarBlue was used to determine IC-50 values of the MDM2 inhibitor AMG-232. Western blot analysis of Cleaved PARP and Annexin V staining for FLOW was used to measure apoptosis and Cyclins A, E, B and p-Histone H3 and flow cytometry to measure cell cycle progression. yH2AX immunofluorescence was used to measure dsDNA breaks.
Results: A MDM2 inhibitor (JNJ-26854165) was nominated as an effective drug in treatment for RT-resistant BC cell lines (R2 = 0.43, p-value <0.01) in our novel radiosensitizer screen. Differential gene expression and pathway analysis in multiple non-overlapping ER+ BC cell lines treated +/-RT identified apoptosis, cell cycle, and p53 signaling as the top pathways induced in ER+ cell lines by RT. Within these MDM2 was significantly overexpressed after RT+ compared to RT- in ER+ p53 wild-type (WT) cells. In p53 mutant (MT) cell lines, however, MDM2 was not differentially expressed. This suggests MDM2 may mediate radioresistance in a p53 dependent manner. Cell growth in the p53 WT cell lines MCF-7 and ZR-75-1 was inhibited by AMG-232, an MDM2 inhibitor (IC-50 values of 554nM and 264nM). p53 MT ER+ cell lines were not sensitive to MDM2 inhibition with this drug (IC-50> 10uM). Clonogenic survival assays demonstrated that at sub-IC50 doses MDM2 inhibition leads to radiosensitization in p53 WT ER+ cell lines (MCF-7 rER: 1.17-2.13; ZR751 rER: 1.30-1.65), however, p53 MT ER+ cells were not radiosensitized (T47D rER: 0.94-1.11; CAMA-1 rER: 0.88-0.95). AMG-232 and RT combined led to an increase in apoptosis compared to RT alone in ER+ p53 WT cells but not p53 MT cells. Combination treatment led to differential cyclin and p-Histone H3 expression in p53 WT cells but not p53 MT cells. G1 cell cycle arrest was a secondary effect of MDM2 inhibition and radiation. Experiments investigating the role of dsDNA breaks in radiosensitization are ongoing.
Conclusions: Our novel radiosensitizer screen identifies MDM2 as a potential mediator of radioresistance in ER+ BC in a p53-dependent manner and suggests that MDM2 targeting concurrent with RT may represent a tractable clinical strategy in women with locally advanced ER+, p53 WT BC.
Citation Format: Cassandra Lynne Ritter, Benjamin C. Chandler, Andrea M. Pesch, Anna R. Michmerhuizen, Nicole Hirsh, Tanner Ward, Amanda Zhang, Mattia Cremona, Lori J. Pierce, Bryan Hennessy, Corey W. Speers. The role of MDM2 inhibition in the radiosensitization of ER+ breast cancers abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1386.
Abstract
Background: Radiation therapy (RT) is a mainstay of treatment for most women with breast cancer (BC). Despite this treatment, response remains heterogenous for women with estrogen receptor ...positive (ER+) BC. Thus, approaches that result in radiosensitization of aggressive ER+ disease are critically needed. We performed a radiosensitizer screen paired with transcriptomic and proteomic data from ER+ models treated +/-RT to identify potential mediators of RT resistance.
Methods: Clonogenic survival assays were used to determine RT sensitivity of 21 BCC lines as well as radiosensitization with drug treatment. IC50 values were determined for 130 clinical compounds and correlation coefficients were calculated using IC50 values and SF-2Gy. Microarray and RPPA data was used for differential gene/protein expression and pathway analysis. AlamarBlue was used to determine IC50 values of the MDM2 inhibitor AMG-232. Western blot analysis of Cleaved PARP was used to measure apoptosis and Cyclins A and E to measure cell cycle progression.
Results: Our radiosensitizer screen nominated the MDM2 inhibitor (JNJ-26854165) as one of the most effective drugs in treating RT-resistant BC cell lines (R2= 0.43, p-value <0.01). In addition, differential gene expression and pathway analysis in multiple non-overlapping ER+ BC cell lines treated +/-RT identified apoptosis, cell cycle, and p53 signaling as the top pathways induced by RT in ER+ cell lines. Within these pathways MDM2 was significantly overexpressed after RT compared to RT- in ER+ p53 wild-type (WT) cells. However, in p53 mutant (MT) cell lines MDM2 was not differentially expressed suggesting MDM2 may mediate radioresistance in a p53 dependent manner. The MDM2 inhibitor AMG-232 inhibited cell growth in the p53 WT cell lines MCF-7 and ZR-75-1 (IC-50 values of 554nM and 264nM, respectively). In contrast, p53 MT ER+ cell lines were not sensitive to MDM2 inhibition (IC-50> 10uM). Clonogenic survival assays demonstrated that MDM2 inhibition at sub-IC50 doses leads to radiosensitization in p53 WT ER+ cell lines (MCF-7 rER: 1.37-1.66;ZR751 rER: 1.30-1.65). In contrast, p53 MT ER+ cells did not demonstrate significant radiosensitization (T47D rER: 0.94-1.11). Combination of AMG-232 and RT led to an increase in apoptosis compared to RT alone in ER+ p53 WT cells but not p53 MT cells. Additionally, combination treatment led to differential cylin expression in p53 WT cells but not p53 MT cells. In vivo studies testing MDM2 inhibition with RT in p53 WT and MT orthotopic and PDX models are ongoing.
Conclusions: Our novel radiosensitizer screen identifies MDM2 as a potential mediator of radioresistance in ER+ BC. Additionally, MDM2 inhibition confers radiosensitization in a p53 dependent manner in ER+ BC and may represent a tractable clinical strategy in women with p53 WT BC.
Citation Format: Cassandra L. Ritter, Benjamin C. Chandler, Andrea M. Pesch, Anna R. Michmerhuizen, Nicole Hirsh, Amanda Zhang, Tanner Ward, Mattia Cremona, Bryan Hennessy, Lori J. Pierce, Corey W. Speers. A radiosensitizer screen identifies a novel role for MDM2 inhibition in the radiosensitization of ER+ breast cancers in a p53 dependent manner abstract. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6270.
Abstract
Purpose: Compared to other breast cancer subtypes, triple negative breast cancers (TNBC) derive the least benefit from adjuvant radiation (RT) which contributes to higher rates of ...locoregional recurrence. Thus, there is a critical need to identify clinical strategies to increase the effectiveness of RT therapy in TNBC.
Methods: Alamar blue proliferation assays were used to calculate half maximal inhibitory concentration (IC50) values for each Bcl-2 family inhibitor 72 hours after drug treatment. Clonogenic survival assays were used to evaluate radiosensitivity and to calculate the radiation enhancement ratio (rER) after combination treatment. Apoptosis was assessed through formation of cleaved PARP and annexin V/PI-based flow cytometry. Xenograft models with MDA-MB-231 cells and TNBC patient-derived xenografts (PDX4664) were used to assess radiosensitization in vivo.
Results: A novel radiosensitizer screen identified Bcl-2 family inhibition as a potentially effective treatment strategy in radioresistant breast cancer models. Single-agent response to pan Bcl-2 family inhibition (ABT-263) or Bcl-xL inhibition (WEHI-539, A-1331852) was more effective in PIK3CA wild type (wt) TNBC (IC50 < 1µM) compared to PIK3CA mutant TNBC. Inhibition of apoptosis with ABT-263 led to radiosensitization of PIK3CA/PTEN wild-type TNBC cell lines (rER: 1.09-1.74), but had no effect on PIK3CA/PTEN mutant TNBC (rER: 0.87-1.18). Radiosensitization was observed to be Bcl-xL-dependent, with Bcl-xL inhibitor-specific radiosensitization (rER: 1.12-2.38) but a lack of Bcl-2 inhibitor (ABT-199, rER: 0.94 - 1.21) or MCL-1 inhibitor-mediated radiosensitization (S63845, rER: 0.91 - 1.06). In PIK3CA wt TNBC, combination treatment of Bcl-2 family inhibition and RT significantly increased the percent of apoptotic cells (p < 0.001) and led to increased formation of cleaved PARP 48 hours after RT. Sensitivity to RT was dependent on expression of MCL-1, an anti-apoptotic protein that is overexpressed in PIK3CA/PTEN mutant TNBC. Overexpression of MCL-1 in PIK3CA/PTEN wild type TNBC rescued radioresistance (rER: 0.99-1.09), whereas co-inhibition of MCL-1 and Bcl-xL in PIK3CA/PTEN mutant TNBC was sufficient to overcome radioresistance (rER: 2.32 - 2.35). In vivo, nonspecific Bcl-2 family inhibition or specific Bcl-xL inhibition in combination with RT decreased tumor growth and increased time to tumor tripling (p < 0.0001) in PIK3CA wt models of TNBC.
Conclusions: In this study, we demonstrated that inhibition of Bcl-2 family proteins in combination with RT led to increased levels of apoptosis and cell death in PIK3CA/PTEN wt - but not PIK3CA/PTEN mutant - TNBC and we identified MCL-1 as a critical mediator of this radiosensitIvity. Together, these results indicate that Bcl-xL inhibition may be a feasible clinical strategy for the radiosensitization of PIK3CA/PTEN wild-type TNBC.
Citation Format: Andrea M. Pesch, Benjamin C. Chandler, Anna R. Michmerhuizen, Nicole Hirsh, Kari Wilder-Romans, Meilan Liu, Tanner Ward, Dana Messinger, Charles Nino, Cassandra Ritter, James M. Rae, Corey W. Speers. Radiosensitization of PIK3CA wild type triple negative breast cancers with Bcl-family inhibition abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1943.
Abstract
Background: Basal-like breast cancer (BC) has the highest rates of local recurrence despite the use of radiation therapy. Therefore, approaches for radiosensitization are critically needed ...for patients with this subtype of BC.
Methods: Four independent datasets were used to correlate gene expression with local recurrence (LR) and Kaplan-Meier analysis validated the impact of TTK expression on LR. The METABRIC dataset was used to determine TTK expression in BC subtypes. Clonogenic survival assays were used to determine the radiosensitization of cell lines after TTK inhibition (TTKi). Mouse models were used to assess TTKi in combination with RT in vivo. DNA damage was quantified using γH2AX staining. HR and NHEJ efficiency assays were performed using HR/NHEJ specific reporter systems. HR competency was also assessed using Rad51 foci formation assays. Rescue experiments were performed using wild-type (WT) and kinase-dead (KD) TTK plasmids in combination with siRNA targeting the UTR region of TTK.
Results: Ten genes were found to significantly correlate with early LR (≤3 years) after surgery and radiation across 4 independent datasets (N=896 pts), with TTK, a cell cycle kinase, ranked the highest. Kaplan-Meier survival analysis in multiple cohorts demonstrated that higher than median TTK expression correlates with decrease LR free survival after RT (HR 1.70-2.42, p<0.01 for all 3 cohorts). Subtype association analysis demonstrated that TTK expression was most elevated in basal-like BC. Using inducible shRNA, the combination of TTK knockdown and RT increases radiosensitivity in multiple basal-like BC cell lines (rER 1.21-1.63). Additionally, TTKi using, Bayer 1161909 (B909), enhanced radiosensitivity in multiple cell lines (rER 1.10-2.27). In vivo, TTKi, using shRNA or B909, in combination with RT led to delayed tumor growth and a significant increase in time to tumor tripling (Placebo: 9 days vs. B909+RT: undefined >35 days, p<0.0001) in both cell line and PDX models. Increased DNA damage was found after combination treatment of TTKi and RT compared to RT alone, indicating that DNA damage repair mechanisms may be compromised by TTKi. The efficiency of the double strand DNA damage repair mechanism, homologous recombination (HR), but not non-homologous end joining (NHEJ), was reduced upon TTKi in HR/NHEJ specific reporter systems. Additionally, Rad51 foci formation was reduced by TTKi after RT compared to RT alone. Reintroduction of WT TTK, after knockdown of endogenous TTK, rescued radioresistance and HR efficiency, however, reintroduction of kinase-dead (KD) TTK was unable to do so in multiple cell lines. WT TTK also rescued Rad51 foci formation after knockdown of endogenous TTK while KD TTK did not.
Conclusion: These data support TTKi as a radiosensitizing strategy for clinical development in basal-like BC patients and that radiosensitization is mediated, at least in part, through impaired HR repair.
Citation Format: Benjamin Chandler, Leah Moubadder, Cassandra Ritter, Meilan Liu, Meleah Cameron, Kari Wilder-Romans, Amanda Zhang, Andrea Pesch, Anna Michmerhuizen, Nicole Hirsh, Marlie Androsiglio, Tanner Ward, Eric Olsen, Yashar Niknafs, Sofia Merajver, Dafydd Thomas, Powel Brown, Theodore Lawrence, Shyam Nyati, Lori Pierce, Arul Chinnaiyan, Corey Speers. TTK inhibition radiosensitizes basal-like breast cancer through impaired homologous recombination abstract. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6273.
Patients with radioresistant breast cancers, including a large percentage of women with triple negative breast cancer (TNBC), demonstrate limited response to radiation (RT) and increased locoregional ...recurrence; thus, strategies to increase the efficacy of RT in TNBC are critically needed. We demonstrate that pan Bcl-2 family inhibition (ABT-263, rER: 1.52-1.56) or Bcl-xL specific inhibition (WEHI-539, A-1331852; rER: 1.31-2.00) radiosensitized wild-type
TNBC (MDA-MB-231, CAL-120) but failed to radiosensitize mutant
TNBC (rER: 0.90 - 1.07; MDA-MB-468, CAL-51, SUM-159). Specific inhibition of Bcl-2 or Mcl-1 did not induce radiosensitization, regardless of
status (rER: 0.95 - 1.07). In wild-type
TNBC, pan Bcl-2 family inhibition or Bcl-xL specific inhibition with RT led to increased levels of apoptosis (p < 0.001) and an increase in cleaved PARP and cleaved caspase 3. CRISPR-mediated
knockout in wild-type
MDA-MB-231 and CAL-120 cells induced expression of pAKT/Akt and Mcl-1 and abolished Bcl-xL inhibitor-mediated radiosensitization (rER: 0.94 - 1.07). Similarly, Mcl-1 overexpression abolished radiosensitization in MDA-MB-231 and CAL-120 cells (rER: 1.02 - 1.04) but transient
knockdown in CAL-51 cells promoted Bcl-xL-inhibitor mediated radiosensitization (rER 2.35 ± 0.05).
, ABT-263 or A-1331852 in combination with RT decreased tumor growth and increased tumor tripling time (p < 0.0001) in
wild-type TNBC cell line and patient-derived xenografts. Collectively, this study provides the preclinical rationale for early phase clinical trials testing the safety, tolerability, and efficacy of Bcl-xL inhibition and RT in women with wild-type
wild-type TNBC at high risk for recurrence.
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