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.
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
> 10 μmol/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
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
, 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 ± PARPi in women with IBC (SWOG 1706, NCT03598257).
Unmet clinical needs in breast cancer (BC) management include the identification of patients at high risk of local failure despite adjuvant radiation and an understanding of the biology of these ...recurrences. We previously reported a radiation response signature and here extend those studies to identify a signature predictive of recurrence timing (before or after 3 years).
Two independent patient cohorts were used. The training cohort included 119 patients with in-breast tumor recurrence (343 total), and the validation testing cohort had 16 patients with recurrences (112 total). All patients received radiation treatment after breast-conserving surgery. Initial feature selection used Spearman rank correlation, and a linear model was trained and locked before testing and validation. Cox regression was used for univariate and multivariable analyses (UVA and MVA, respectively). Biologically related concepts were identified using gene set enrichment analysis.
Spearman correlation identified 485 genes whose expression was significantly associated with recurrence time (early vs late). Feature reduction further refined the list to 41 genes retained within the signature. In training, the correlation of score to recurrence time was 0.85 (P value < 1.3 × 10
) with an area under the curve (AUC) of 0.91. Application of this early versus late signature to an independent BC testing and validation set accurately identified patients with early versus late recurrences (Spearman correlation = 0.75, P value = .001, AUC = 0.92, sensitivity = 0.75, specificity = 1.0, positive predictive value = 1.0, and negative predictive value = 0.8). Unique associations of breast cancer intrinsic subtype to timing of local recurrence were identified. In UVA and MVA the early versus late recurrence signature remained the most significant factor associated with recurrence. Gene set enrichment analysis identified proliferation and epidermal growth factor receptor concepts associated with early recurrences and luminal and ER-signaling pathways associated with late recurrences. Knockdown of genes associated with the early and late recurrences demonstrated novel effects on proliferation and clonogenic survival, respectively.
We report a breast cancer gene signature that may identify patients unlikely to respond to adjuvant radiation and may be used to predict timing of recurrences with implications for potential treatment intensification and duration of follow-up for women with breast cancer treated with radiation.
Abstract
Background: Increased rates of locoregional recurrence have been observed in basal-like breast cancer despite the use of radiation therapy (RT), therefore approaches that result in ...radiosensitization of basal-like breast cancer are critically needed. Studies detailing the poor response of basal-like tumors to adjuvant RT underscore the biologic differences and as of yet undefined oncogenic drivers of these particular types of breast cancer.
Methods: 4 independent datasets were used to correlate gene expression with local recurrence (LR) after 3 years. Kaplan-Meier analysis was used to validate the impact of TTK expression on LR. The TCGA and institutional breast cancer dataset were used to determine TTK expression in BC subtypes. TTK RNA and protein levels were measured using qPCR and western blot at baseline and correlated to intrinsic radiosensitivity. Clonogenic survival assays were used to determine the radiosensitization of cell lines after TTK inhibition (TTKi). Mice 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.
Results: Ten genes were found to significantly correlate with early LR (≤3 years) across 4 distinct datasets (N=896 pts) (OR of recurrence > 2, p-value <0.000001), with TTK, a cell cycle kinase, ranked the highest. Kaplan-Meier survival analysis in 3 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 breast cancer (p<0.0001). Clonogenic survival assays, using inducible shRNA models, show the combination of TTK knockdown and RT increases radiosensitivity in MDA-MB-231 and BT-549 basal-like breast cancer cell lines (rER 1.21-1.63). Additionally, TTKi using, Bayer 1161909 (B909), enhanced radiosensitivity in both cell lines (rER 1.10-1.39). In vivo, TTKi, using shRNA and B909 in combination with RT led to a significant increased time to tumor tripling. Using γH2AX foci formation assays, 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. We found that 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.
Conclusion: These data support TTK inhibition as a rationale radiosensitizing strategy for clinical development in basal-like breast cancer patients and that the mechanism of radiosensitization is, at least in part, through impaired HR repair.
Citation Format: Benjamin C. Chandler, Leah Moubadder, Cassie Ritter, Kari Wilder-Romans, Meleah Cameron, Meilan Liu, Shyam Nyati, Andrea Pesch, Anna Michmerhuizen, Eric Olsen, Yashar Niknafs, Arul Chinnaiyan, Corey Speers. Nomination and characterization of TTK for radiosensitization in basal-like breast cancers 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 2926.
Abstract
Purpose: Sustained locoregional control of disease is a significant issue in patients with inflammatory breast cancer (IBC) with local control rates <70% at 5 years in these patients. Given ...the unsatisfactory outcomes, there is a clear need for intensification of local therapy, including radiation, for these patients. Inhibition of poly(adenosine diphosphate-ribose) polymerase 1 (PARP1), a DNA repair protein, represents a novel and promising strategy for radiosensitizing aggressive tumors. The purpose of this study was to investigate radiosensitization by PARP1 inhibition in IBC and to determine the mechanism of radiosensitization.
Methods: Pharmacologic PARP1 inhibitors, olaparib and veliparib, were used at various concentrations (10-5 to 10-12 M). Proliferation assays were used to determine the IC50s of these inhibitors in three independent IBC cell lines (SUM 149, SUM 190, MDA-IBC-3). Clonogenic survival assays were used to determine the radiosensitization in cell lines after pharmacologic PARP inhibition (PARPi). DNA damage was quantified using γH2AX staining in IBC cell lines. Transcriptomic, protein and signaling pathway changes were measured using RNA-Seq and RPPA, respectively. In vivo tumor growth was also measured in CB17-SCID mice with varying control and treatment groups (16-20 tumors/group) that included radiation (RT) alone, olaparib alone, and RT+olaparib.
Results: PARPi via olaparib and veliparib had limited single agent efficacy in all three independent IBC models with IC50 values >10 µM. Despite limited single agent efficacy, sub micromolar concentrations of PARPi with RT led to significant radiosensitization with radiation enhancement ratios of 1.20-1.35 in the same three models (500 nM in SUM 190, 500 nM in MDA-IBC-3, 2 nM in SUM 149). This effect was partially dependent on BRCA 1/2 mutational status. Radiosensitization was due, at least in part, to delayed resolution of double strand DNA (dsDNA) breaks at 12, 16, and 24 hours in all models. Additionally, this effect is mediated, at least in part, by a non-homologous end joining (NHEJ) mechanism and was not dependent on homologous recombination (HR). RNA-Seq and RPPA protein pathway data identify key pathways that regulate the radiosensitization in these IBC models. Additionally, xenograft studies show significant differences in tumor doubling and tripling times between treated and untreated tumors.
Conclusion: Our study demonstrates that the PARP1 inhibition improves the therapeutic index of radiotherapy in IBC cell lines. Combined PARPi with RT leads to unresolved dsDNA breaks and decreased clonogen survival. These studies provide the rationale for a phase II randomized trial of RT +/- PARPi (olaparib) in women with IBC that has recently opened for accrual (SWOG 1706 - NCT03598257). This work was supported by a grant from Komen for the Cure and NIH T-32-GM007315.
Citation Format: Anna Michmerhuizen, Andrea Pesch, Leah Moubadder, Meleah Cameron, Amanda Zhang, Nicole Hirsh, Meilan Liu, Kari Wilder-Romans, Lori J. Pierce, Reshma Jagsi, Corey Speers. PARP inhibition as a radiosensitizing strategy to improve locoregional control in inflammatory breast cancer 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 3924.
Abstract
Background: Increased rates of locoregional recurrence leading to poorer clinical outcomes have been observed in triple-negative breast cancer (TNBC) despite the use of radiation therapy ...(RT), therefore approaches that result in radiosensitizaton in TNBC are critically needed. Our previous work identified a group of cell cycle kinases differentially expressed in estrogen receptor negative (ER-) versus estrogen receptor positive (ER+) breast cancer (BC). Here we described the impact of elevated expression of one of these identified kinases, TTK, on radiation response and patient outcomes in TNBC.
Methods: TCGA breast cancer datasets were used to determine TTK expression in the intrinsic subtypes of BC. TTK RNA and protein levels were measured using qPCR and western blot at baseline and after radiation treatment. Clonogenic survival assays were used to determine the radiosensitization in several cells after TTK inhibition. DNA damage was quantified using γH2AX staining in TNBC cell lines. Kaplan-Meier analysis was used to determine the impact of TTK expression on locoregional recurrence (LRR) and overall survival (OS). A Cox proportional hazards model was constructed to identify potential factors of LRR-free survival in univariate (UVA) and multivariable analyses (MVA).
Results: TTK expression is elevated in breast cancer tissue compared to normal breast tissue (Q-VAL: 8.70 E-291) and is most highly expressed in basal-like tumors. TTK is overexpressed in ER- versus ER+ tumors in both breast cancers patient samples (p-value: <0.0001), and cells lines (p-value: <0.0001). TTK expression is significantly correlated with intrinsic radioresistance in a panel of 23 BC cell lines (R=0.58, p-value=0.0035). TTK RNA and protein levels are significantly increased at 12 and 24 hours after RT in TNBC cell lines. Genomic (siRNA knockdown) or pharmacologic (NMS-P715) inhibition of TTK increased radiosensitivity in vitro in 3 different TNBC cell lines. This sensitization is mediated, at least in part, through impaired DNA damage repair. Increased γH2AX foci was found after combination treatment of RT and TTK inhibition compared to only TTK inhibition, or RT. Clinically, breast cancer patients treated with breast conserving surgery and RT whose tumors have higher than median expression of TTK had worse local-recurrence free (LRF) survival and overall survival (HR for local recurrence 1.7 as continuous variable, p-value 0.004) compared to patients with higher than median expression, and this pattern held when considering quartile expression. TTK expression was associated with poorer LRF survival in UVA and in MVA only TTK expression and grade were significantly associated with worse LRF survival in 3 independent datasets.
Conclusion: Our results support the rationale for developing translational clinical trials to investigate TTK inhibition as a novel radiosensitizing target in TNBC.
Citation Format: Benjamin C. Chandler, Leah Moubadder, Cassie Ritter, Yashar Niknafs, Eric Olsen, Meleah Cameron, Meilan Liu, Kari Wilder-Romans, Shyam Nyati, Powel Brown, Arul Chinnaiyan, Corey Speers. TTK: A novel target for radiosensitization in triple-negative breast cancers abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 677.
Abstract
Background: Despite previous studies suggesting that MELK is not involved in proliferation in cancer, many groups have shown that MELK is a critical mediator of radioresistance and may be an ...ideal target for radiosensitization. Furthermore, additional studies suggest MELK functions to regulate the cell-cycle and that its mRNA and protein levels are regulated during cell-cycle, although the mechanisms of this regulation are currently unknown. We sought to identify mediators of MELK protein regulation, determine whether it was ubiquitin-ligase mediated, and if so, which E3-ubiquitin ligase machinery degrades MELK during cell-cycle progression.
Methods: Sequence alignments were carried out to identify degron motifs in MELK. Site directed mutagenesis were performed to mutate degrons in MELK. CDC20 and CDH1 were over-expressed to identify the co-activators of the E3-ubiquitin ligase complex. Cells were synchronized by double thymidine block or by nocodazole. CDC27/APC3 and CDC20 antibodies were used to pull-down the Anaphase Promoting Complex/Cyclosome (APC/C) and Mitotic Checkpoint Complex (MCC), respectively. Resulting pellets were probed with antibodies against BubR1/Mad3, Bub3, CDC20, CDC27, MELK, Cyclin B1, CDK1, BUB1, and Mad2. A role for MELK kinase activity on its half-life and formation of APC/C and MCC was evaluated using kinetically inactive (MELK-KD) mutant or OTSSP167. siRNA against MELK was used to confirm a requirement of MELK for formation of APC/C and MCC. The effect of MELK degron mutant on radiosensitization will be evaluated in MELK CRISPR TNBC cell lines.
Results: In this study we provide evidence that MELK has two D-boxes and a KEN box which are largely conserved from C. elegance to H. sapiens. We identified that MELK is degraded by the E3-ubiquitin ligase APC/C with the coactivator being CDC20. Rescue with MG132 confirms that MELK is degraded by a proteosomal-mediated pathway. CO-IP studies show that MELK is required for APC/C and MCC formation and that MELK interacts with CDC20, CDK1, BUB1, and BUBR1. Cycloheximide chase studies show D-Box1 and KEN box mutants of MELK are more stable while D-BOX2 mutants are least stable. The KEN and D-Box mutant MELK show impairment in APC/C and MCC formation, altered Cyclin B1-CDK1 and Cyclin B1-CDC20 interactions. MELK KD and OTSSP prove that kinase activity of MELK plays a role in its stability. We anticipate that the least stable MELK mutant will result in higher radiosensitivity compared to MELK-WT.
Conclusions: MELK is regulated by APC/CCDC20 through its D-Box1, KEN and D-Box2. Degron mutant and MELK KD show altered protein stability which leads to attenuated APC/C and MCC formation. These results provide proof that MELK is a cell-cycle checkpoint kinase and support the rationale for developing clinical strategies to degrade and inhibit MELK in combination with radiation therapy.
Citation Format: Shyam Nyati, Benjamin Chandler, Anna Michmerhuizen, Andrea Pesch, Cassandra Ritter, Leah Moubadder, Meilan Liu, Meleah Cameron, Eric Olsson, Kari Wilder-Romans, Dipankar Ray, Theodore S. Lawrence, Felix Y. Feng, Lori J. Pierce, Corey Speers. Discovery of degradation pathway for maternal embryonic leucine zipper kinase (MELK): Implications for breast cancer therapy 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 2539.
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.
Abstract
Background: Increased rates of locoregional recurrence have been observed in TNBC despite the use of radiation (RT); therefore, approaches that result in radiosensitizaton in TNBC may ...improve clinical outcomes. Despite some controversy as to the role of MELK kinase as an oncogene, we previously established its clear role in conferring radioresistance in TNBC. Here we extend those studies to understand the mechanism of conferred radioresistance.
Methods: Clonogenic survival assays were used to quantify the degree of radiosensitivity after MELK inhibition. Mass spectrometry was utilized to identify proteins associated with MELK. NHEJ assays were performed using linearized pEYFP vector and quantitated by FACS. qRT-PCR was used to determine MELK RNA expression. Rad51 and γH2AX foci counting were performed to evaluate homologous recombination (HR). Neutral comet assay was used to quantitate dsDNA break repair. Laser microirradiation studies were performed to determine the location and kinetics of protein shuttle to and from sites of dsDNA breaks, including MELK, Ku70 and Ku80 proteins. Fluorescent microscopy was performed for localization of endogenous MELK protein post RT. To study the effect of MELK kinase activity on Ku70-Ku80 complex formation and recruitment at the DNA damage site, cells were lysed and immunoprecipitation was performed using MELK, Ku70 or Ku80-specific antibodies.
Results: We previously demonstrated that genomic or pharmacologic inhibition of MELK confers significant radiosensitization. Mechanistic studies were undertaken to understand the pathways critical to MELK-mediated radioresistance. When MELK is genomically or pharmacologically inhibited, NHEJ and HR reporter assays demonstrate that this radiosensitization is driven through impaired NHEJ at 4, 16, and 24 hrs after RT and not mediated by HR in multiple TNBC cell lines. Tandem mass spectrometry studies of tagged MELK protein identify Ku70 and Ku80 as direct interactors with MELK protein, which is confirmed by Co-IP. Laser microirradiation studies confirm that MELK, Ku70 and Ku80 co-localize to sites of dsDNA breaks, and that MELK kinase function is required to stabilize the Ku70/Ku80 complex at these sites. Comet assay confirms that rapid dissolution of the Ku70/Ku80 complex when MELK is inhibited or functionally dead is not a result of accelerated dsDNA break repair.
Conclusion: MELK kinase function is critical in conferring radioresistance in TNBC and inhibition of function confers radiosensitivity through an NHEJ-mediated pathway. MELK also stabilizes Ku70/Ku80 proteins at sites of dsDNA breaks and allows for more efficient repair of breaks induced by ionizing RT. These results further support the rationale for developing clinical strategies to inhibit MELK in combination with RT treatment as a novel radiosensitizing strategy in TNBC.
Citation Format: Shyam Nyati, Ben Chandler, Eric Olsen, Leah Moubadder, Meilan Liu, Meleah Cameron, Kari Wilder-Romans, Theodore S. Lawrence, Powel H. Brown, Fellix Y. Fang, Lori J. Pierce, Corey Speers. Maternal embryonic leucine zipper kinase (MELK) confers radioresistance in triple-negative breast cancers (TNBC) through a nonhomologous end joining (NHEJ)-mediated pathway abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3218.
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