-Because it leads to a rapid and massive muscle hypertrophy, postnatal blockade of the activin type IIB receptor (ActRIIB) is a promising therapeutic strategy for counteracting muscle wasting. However, the functional consequences remain very poorly documented in vivo. Here, we have investigated the impact of 8-wk ActRIIB blockade with soluble receptor (sActRIIB-Fc) on gastrocnemius muscle anatomy, energy metabolism, and force-generating capacity in wild-type mice, using totally noninvasive magnetic resonance imaging (MRI) and dynamic 31 P-MRS. Compared with vehicle (PBS) control, sActRIIB-Fc treatment resulted in a dramatic increase in body weight (ϩ29%) and muscle volume (ϩ58%) calculated from hindlimb MR imaging, but did not alter fiber type distribution determined via myosin heavy chain isoform analysis. In resting muscle, sActRIIB-Fc treatment induced acidosis and PCr depletion, thereby suggesting reduced tissue oxygenation. During an in vivo fatiguing exercise (6-min repeated maximal isometric contraction electrically induced at 1.7 Hz), maximal and total absolute forces were larger in sActRIIB-Fc treated animals (ϩ26 and ϩ12%, respectively), whereas specific force and fatigue resistance were lower (Ϫ30 and Ϫ37%, respectively). Treatment with sActRIIB-Fc further decreased the maximal rate of oxidative ATP synthesis (Ϫ42%) and the oxidative capacity (Ϫ34%), but did not alter the bioenergetics status in contracting muscle. Our findings demonstrate in vivo that sActRIIB-Fc treatment increases absolute force-generating capacity and reduces mitochondrial function in glycolytic gastrocnemius muscle, but this reduction does not compromise energy status during sustained activity. Overall, these data support the clinical interest of postnatal ActRIIB blockade. skeletal muscle hypertrophy; activin type IIB receptor; myostatin; force; fatigue ACTIVIN TYPE IIB RECEPTOR (ActRIIB) is a transmembrane serinethreonine kinase receptor highly expressed in mammalian skeletal muscle. It mediates signaling for myostatin (GDF8) and other members of the transforming growth factor-␤ family that are involved in the negative regulation of skeletal muscle development (16,37). Disruption of the ActRIIB signaling pathway leads to a rapid and massive increase in muscle mass resulting from fiber hypertrophy with increased myofiber protein synthesis (10, 58). In that context, several pharmacological approaches based on the postnatal ActRIIB signaling blockade have been considered for counteracting muscle wasting commonly associated to aging, neuromuscular disorders and various catabolic diseases (17,33,38). These approaches mainly include the systemic delivery of neutralizing antibodies against ActRIIB or myostatin (31, 36), and injection of a soluble recombinant form of ActRIIB (sActRIIB-Fc), which acts as a decoy receptor disrupting the interaction of endogenous ActRIIB receptor with its ligands (5,38,44).Although ActRIIB signaling blockade has been already tested in patients (30, 57) and animal models (10,44,47) suffering from dystrophi...

Postnatal blockade of the activin type IIB receptor (ActRIIB) represents a promising therapeutic strategy for counteracting dystrophic muscle wasting. However, its impact on muscle function and bioenergetics remains poorly documented in physiologic conditions. We have investigated totally noninvasively the effect of 8-wk administration of either soluble ActRIIB signaling inhibitor (sActRIIB-Fc) or vehicle PBS (control) on gastrocnemius muscle force-generating capacity, energy metabolism, and anatomy in dystrophic mdx mice using magnetic resonance (MR) imaging and dynamic [P]-MR spectroscopy ([P]-MRS) in vivo ActRIIB inhibition increased muscle volume (+33%) without changing fiber-type distribution, and increased basal animal oxygen consumption (+22%) and energy expenditure (+23%). During an in vivo standardized fatiguing exercise, maximum and total absolute contractile forces were larger (+40 and 24%, respectively) in sActRIIB-Fc treated animals, whereas specific force-generating capacity and fatigue resistance remained unaffected. Furthermore, sActRIIB-Fc administration did not alter metabolic fluxes, ATP homeostasis, or contractile efficiency during the fatiguing bout of exercise, although it dramatically reduced the intrinsic mitochondrial capacity for producing ATP. Overall, sActRIIB-Fc treatment increased muscle mass and strength without altering the fundamental weakness characteristic of dystrophic mdx muscle. These data support the clinical interest of ActRIIB blockade for reversing dystrophic muscle wasting.-Béchir, N., Pecchi, E., Vilmen, C., Le Fur, Y., Amthor, H., Bernard, M., Bendahan, D., Giannesini, B. ActRIIB blockade increases force-generating capacity and preserves energy supply in exercising mdx mouse muscle in vivo.

Background Patients with triple-negative breast cancer (TNBC) develop early recurrence. While PARP inhibitors (PARPi) have demonstrated potential in BRCA1/2-mutant (BRCAMUT) TNBC, durable responses will likely be achieved if PARPi are used in combination. It is plausible that sequential administration of a potent PARPi like talazoparib in combination with carboplatin can enhance primary tumour and metastasis inhibition in BRCAMUT and BRCA1/2 wild-type (BRCAWT) TNBCs, and decrease toxicity. Methods We evaluated the impact of the concurrent combination of talazoparib and carboplatin on cell survival in 13 TNBC cell lines. We compared the concurrent and sequential combination upon fork replication, migration and invasion. We also used three orthotopic xenograft models to evaluate primary tumour growth, distant metastasis, and toxicity. Results Concurrent talazoparib and carboplatin was synergistic in 92.3% of TNBC cell lines, independent of BRCA1/2-mutation status. The sequential combination decreased fork speed in normal cells, but not in TNBC cells. The talazoparib-first sequential combination resulted in a strong reduction in migration (70.4%, P < 0.0001), invasion (56.9%, P < 0.0001), lung micrometastasis (56.4%, P < 0.0001), and less toxicity in a BRCAWT model. Conclusion The sequential combination of talazoparib and carboplatin is an effective approach to inhibit micrometastatic disease, providing rationale for the use of this combination in early TNBC patients.

586 Background: PARP inhibitors (PARPi) such as talazoparib and olaparib, have demonstrated an improvement in progression-free survival (PFS) amongst metastatic HER2-negative breast cancer patients with germline mutations in BRCA1/2 (BRCA-MUT). Clinical trials have evaluated PARPi in combination with carboplatin, but with mixed results. Earlier trials studied the combination of carboplatin and a low-dose PARPi of low potency, veliparib. The concomitant combination of carboplatin and talazoparib, a higher-potency PARPi, was also evaluated in solid tumors, using a heavily pre-treated population. Here, we perform a comparative evaluation of different sequencing strategies of talazoparib and carboplatin to determine efficacy and toxicity in BRCA-MUT and BRCA wild-type (WT) TNBC models. Methods: We used three orthotopic xenograft models in NSG (NOD scid gamma) mice, with 7-14 mice in each treatment group: MDAMB231 (BRCA-WT), HCC1806 (BRCA-WT), and MX1 (BRCA-MUT). We treated mice with carboplatin (C) (35 mg/kg intraperitoneally) in combination with talazoparib (T) (0.33 mg/kg oral gavage) using 2 dosing strategies: a) concomitant administration of C + T; and b) T first, followed by C three days later, each compared to vehicle control. We evaluated primary tumor inhibition and hematologic toxicity. Kruskal-Wallis test and Dunn’s multiple comparison test was used to assess statistical significance. Results: Using the MDAMB231 xenograft, we found the T-first approach led to a 66.7% (P < 0.0001), and the concomitant approach resulted in a 51.4% decrease in primary tumor volume, (P = 0.08), in comparison to control. In HCC1806, the T-first approach resulted in a 62.0% decrease in tumor volume (P < 0.0001), whereas the concomitant combination showed a 54.4% decrease (P = 0.002). In MX1, the T-first and concomitant approaches resulted in 72.7% (P < 0.0001) and 81.4% (P < 0.0001) decrease in tumor volume, respectively. With regards to neutrophil counts, T-first approach decreased neutrophils by 66.2% and 43.0% in MDAMB231 and HCC1806 xenografts respectively, similar to the trend with concomitant T + C: 61.4% and 38.0%. In the MX1 cohort, the T-first approach resulted in a 66.2% decrease in neutrophils (P = 0.001), and the concomitant approach led to a 77.5% decrease in neutrophils (P = 0.006). Conclusions: Our results demonstrate that the talazoparib-first approach is effective in two BRCA-WT models with no statistically significant neutropenia. While the concomitant combination approach demonstrated greater tumor inhibition in the BRCA-MUT model, this was also associated with significant neutropenia. This is suggestive that sequencing of talazoparib and carboplatin may have differential effect in BRCA-WT and BRCA-MUT tumors and may play an important role in improving efficacy in BRCA-WT tumors.

These findings demonstrate that the increased fatigability following ActRIIB blockade is not due to limitation in energy supply and/or disturbance in contractile ATP cost. This article is protected by copyright. All rights reserved.

Background: PARP inhibitors, such as talazoparib, demonstrated an improvement in progression-free survival as monotherapy in germline BRCA1/2-mutated (BRCA-MUT) HER2-negative locally advanced/metastatic breast cancer patients. BRCA-MUT tumors constitute 15-20% of triple-negative breast cancers (TNBCs). TNBCs are an aggressive subtype and lack overexpression of hormone receptors and HER2. Here, we evaluate the impact of the combination of talazoparib and a chemotherapeutic agent, carboplatin, to better understand which TNBCs will benefit from this combination, and the manner in which different sequencing strategies can impact primary tumor growth and distant metastasis development. Methods: We performed a 10-day chemosensitivity assay using 7 BRCA-MUT and BRCA wild-type TNBC cell lines. We tested 9 concentrations of talazoparib (Pfizer), and carboplatin (Selleckchem) each. Post-treatment, we used automated high-content imaging with Operetta (Perkin Elmer). We calculated IC50 values for talazoparib alone, CI (Combination Index) values, the DNA damage response (product of the percentage of cells positive for 53BP1 and mean 53BP1 foci per cell), and the apoptotic index (percentage of cells positive for cleaved-PARP). We also used an orthotopic xenograft model of MDAMB231 in NSG (NOD scid gamma) mice, with 8-14 mice in each treatment group. We treated mice with carboplatin (C) (35 mg/kg intraperitoneally) in combination with talazoparib (T) (0.03 mg/kg oral gavage) using 2 dosing strategies: a) concomitant administration of C + T; and b) T first, followed by C three days later, each compared to vehicle control. Results: We found a synergistic effect of the combination of talazoparib and carboplatin (CI &lt; 1) in all 7 cell lines, with the greatest synergy (CI&lt; 0.65) identified in three cell lines that were PARPi (PARP inhibitor)-resistant, namely HCC1143, MDAMB231, and Hs578T. For these three cell lines, we found that the mean 53BP1 product score increased by 7-16 fold in combination versus talazoparib alone at 0.2 µM, and the apoptotic index also increased by 4-26 fold at the same conditions. Concomitant administration of talazoparib and carboplatin in the MDAMB231 xenograft resulted in a 53.1% inhibition in primary tumor volume in comparison to control (P=0.0004), whereas sequential administration resulted in a 69.2% primary tumor volume inhibition (P&lt;0.0001). The talazoparib first combination approach also resulted in a 53.9% decrease in lung micrometastasis (P=0.0003). Conclusion: The combination of talazoparib and carboplatin demonstrated a synergistic effect in 7 TNBC cell lines. We found greater DNA damage and cell death amongst PARPi-resistant cell lines and at lower concentrations of talazoparib when combined with carboplatin. In-vivo, sequential administration of talazoparib and carboplatin was the most effective approach to inhibit primary tumor growth. Citation Format: Alexia Cotte, Michèle Beniey, Takrima Haque, Nelly Béchir, Audrey Hubert, Korotoum W. Diallo, Danh Tran-Thanh, Saima N. Hassan. Pre-clinical combination of a PARP inhibitor, talazoparib, and carboplatin in triple-negative breast cancer [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 1066.

Triple-negative breast cancer (TNBC) is a difficult-to-treat breast cancer with limited therapeutic options. PARP inhibitors (PARPi) have emerged as a promising targeted therapeutic for TNBC patients with germline mutations in BRCA1/2, recently demonstrating inhibition of micrometastatic disease. However, studies have also suggested that PARPi may have efficacy in TNBC, regardless of BRCA mutation status. We have previously identified a 63-gene signature associated with the DNA damage response to PARPi, with an overall accuracy of 86% in a cohort of patient-derived xenografts and a predicted sensitivity to PARPi in 45% of untreated TNBC patients. Additionally, our 63-gene signature can be used to identify genes implicated in intrinsic resistance. Therefore, we hypothesize that genes from our 63-gene signature can be used to identify potential targets for combination therapies with PARPi. We selected six candidate genes from our 63-gene signature: BARD1, BUB1, FEN1, RRM2, EXO1, and USP1. For each of the selected genes, we performed siRNA knockdown experiments in MDAMB231, a TNBC cell line. Using flow cytometry, we found that talazoparib, a potent PARPi plus siBARD1, increased the proportion of cells in G2 phase, with 80% γ-H2AX-positive cells and 60% cleaved-caspase-3-positive cells. Interestingly, the DNA damage response was comparable to what was observed with the combination of talazoparib and carboplatin. Talazoparib + siBUB1 also induced DNA damage in 51% of cells (P&lt;0.001), and apoptosis in 20% of cells (P=0.002). Similar effects for enhanced DNA damage and apoptosis with talazoparib was observed for siUSP1, siFEN1, and siEXO1. Furthermore, the combination of siBARD1 + talazoparib resulted in a 49% reduction in cell migration in comparison to siBARD1 alone (P=0.0008), and the combination of siBUB1 and talazoparib demonstrated a 44% reduction in cell migration in comparison to talazoparib alone (P=0.04). Moreover, we evaluated gene expression in different breast cancer subtypes, and correlated with prognosis in a cohort of 881 untreated breast cancer patients. We found elevated expression of RRM2, FEN1, and EXO1 amongst more aggressive breast cancer subtypes (basal and HER2), and that their overexpression were associated with a poorer 10-year distant metastasis-free survival (RRM2, P&lt;0.00001; FEN1, P=0.00012; EXO1, P&lt;0.00001). Taken together, genes from our 63-gene signature can be used to identify therapeutic targets that have demonstrated either enhanced DNA damage, cell death, or cell migration in combination with PARPi. With prognostic implications, these targets also demonstrate therapeutic potential in the clinic and warrant further investigation. Citation Format: Audrey Hubert, Alexia Cotte, Nelly Bechir, Takrima Haque, Saima N. Hassan. Identifying therapeutic targets in combination with PARP inhibitors using a 63-gene signature in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5342.