Takchi, Andrew

Objectives: Developmental Dysplasia of the Hip (DDH) is a common congenital anomaly requiring early diagnosis to minimize invasive treatments. This study evaluates Lebanese pediatricians’ knowledge of DDH risk factors, screening methods, and treatment strategies, comparing their practices to American and European guidelines to propose screening recommendations. Methods: A cross-sectional descriptive study was conducted using a confidential questionnaire to assess Lebanese pediatricians’ knowledge and practices regarding DDH. Contact information for pediatricians was obtained from the Lebanese Order of Physicians. Out of 600 emailed and 100 distributed questionnaires, 263 responses were received, and data were analyzed using proportion tests. Results: Of the 263 respondents (19.9% response rate), 84% routinely ordered radiographs at 4 months, while only 0.4% followed recommended guidelines. About 50% performed hip stability tests (Barlow and Ortolani) at 6 months, and 78.32% recommended the Pavlik harness for post-6-month treatment. Additionally, 97% valued periodic reminders about DDH. Significant gaps were identified in training on risk factor identification, physical examination procedures, imaging indications, management of abnormal findings, and treatment choices. Conclusions: The study highlights a critical training gap in DDH screening among Lebanese pediatricians. Implementing routine neonatal screening aligned with international recommendations is essential to reduce delayed diagnosis and its associated economic burden. Improved education and training on DDH screening and treatment are crucial for early detection and better patient outcomes.

Abstract Background: Despite recent advances in the treatment of triple negative breast cancer (TNBC) many patients are still suffering from early recurrence, therapy resistance, and organ metastasis 1 to 3 years post-chemotherapy. TNBC cells with stem cell-like properties are resistant to standard of care chemotherapy and are responsible for early tumor relapse and poor outcome. Up-regulation of PD-L1 expression in Cancer Stem Cells (CSCs) has been associated with immune evasion capacity and promotion of their stem-like properties. In this study we identify two druggable stemness target, Aurora-A Kinase (AURKA) and NOTCH3, with positive feedback on intra-tumoral PD-L1 expression and cancer cell plasticity. Methods: MDA-MB 231, MDA-MB 231 LM and SUM149-PT TNBC cells were used both in 2D and 3D environment. Unique TNBC cells were established from metastatic PDX models. TNBC cells were treated with Alisertib (AURKA inhibitor, 50nM) or AV353 (NOTCH3 inhibitor, 200ng). Immunofluorescence and immunoblot were used to compare different biomarker expression. ALDH was measured using Aldefluor kit. Apoptotic cells were quantified in real-time using IncuCyte S3. In vivo studies; 1x106 MDA MB 231/LM cells were injected into the mammary fat pad of NSG-female mice and treated with Alisertib or AV353. Control groups were treated with pacebo. Results: Our study shows that AURKA and NOTCH3 pharmacological blockade reduced nuclear/undruggable PD-L1, that is not achieved clinically by using FDA-approved immune checkpoint inhibitors (ICIs). Significantly, following stemness-targeted therapy with alisertib or AV353, reduction of intra-tumoral PD-L1 resulted in inhibition of tumor growth, decreased levels of CD44 expression, increased CD8+ T-cell tumor infiltration, impairment in the enrichment of ALDHhigh CSCs and enhanced chemosensitivity. In vivo pharmacological blockade of AURKA and NOTCH3 signaling pathways induced inhibition of tumor growth, apoptosis, and impairment of organ metastases. Conclusions: This study demonstrates that selective AURKA and NOTCH3 pharmacological blockade results in the inhibition of cancer cell plasticity that is linked to reduction of undruggable nuclear PD-L1 in TNBC cells. Our findings provide the strong preclinical rationale to develop novel clinical trials for patients with advanced TNBC that are refractory to chemotherapy and show limited response to FDA-approved ICIs. Citation Format: Antonino Bonaventura D'Assoro, Matthew Goetz, Carol Lange, Roberto Leon-Ferre, Tufia Haddad, James Ingle, Annapaola Montagner, Andrew Takchi, Ellie Babadi, Lucio Miele, Urban Lendahl, Urban Lendahl. Pharmacological blockade of AURKA and NOTCH3 oncogenic pathways to inhibit cancer cell plasticity in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5978.

e13153 Background: Aurora-A mitotic kinase (AURKA) plays a role in Triple Negative Breast Cancer (TNBC) progression through activation of epithelial to mesenchymal transition (EMT)-mediated cancer cell plasticity. Cancer cells that undergo EMT acquire a CD44high/CD24low and/or ALDHhigh cancer stem-like phenotype (Cancer Stem Cells) characterized by intrinsic drug resistance. Cancer Stem Cells (CSCs) shows high PD-L1 expression that induces tumor immune escape through CD8+ T-cells exhaustion. Significantly, “undraggable” nuclear PD-L1 may play a key role in induction of immune therapy resistance and cancer cell plasticity. Methods: (1) TNBC Models: MDA-MB 231, unique MDA-MB 231/LM Durvalumab resistant (DUV-R) and TNBC-M40 cells established from metastatic PDXs; (2) In Vitro Studies: TNBC cells were treated with the AURKA inhibitor alisertib (50nM). PD-L1 expression was evaluated by immunoblotting. Apoptosis was measured by immunofluorescence using Cleaved-PARP antibodies. To assess stemness capacity, TNBC cells were cultured under non-adherent conditions to form mammospheres. ALDH activity (functional stemness marker) was measured using the Aldefluor Kit. (3) In Vivo Studies: 1x106 MDA-MB 231/LM cells were injected into the mammary fat pad of humanized NSG-CD34+ female mice. Animals were treated with anti-PD-L1 ICI atezolizumab (20 mg/Kg) and alisertib (25 mg/Kg) as monotherapy or in combination. 1x106 MDA-MB 231/LM cells were also injected into the mammary fat pad of humanized NSG-CD34+ female mice and treated with durvalumab (20 mg/Kg) to develop durvalumab-resistant models. PD-L1, CD44, Vimentin expression and tumor infiltration of CD8+ T-cells were assessed by immunofluorescence. Results: Treatment of MDA-MB 231/LM xenografts with alisertib decreased the levels of CD44 and PD-L1 expression and increased CD8+ T-cells tumor infiltration. Because PD-L1 genetic targeting enhanced in vitro alisertib-induced apoptosis, MDA-MB 231/LM xenografts were treated with alisertib and the anti-PD-L1 ICI atezolizumab. Com- bination of atezolizumab and alisertib resulted in the reduced expression of the EMT marker vimentin that was linked to lack of organ metastasis. Combination of atezolizumab and alisertib also induced a significant reduction of ALDHHigh CSC in ex-vivo 3D-Organoids established from TNBC-M40 cells. MDA-MB 231/LM DUV-R cells showed increased nuclear PD-L1 compared to parental cells. The combination of alisertib with Immune Checkpoint Inhibitors (ICIs) induced apoptosis of MDA-MB 231/LM DUV-R cells through down-regulation of intra-tumoral nuclear PD-L1 expression. Conclusions: This study provides innovative pre-clinical rationale for combining AURKA inhibitors with ICIs to reduce “ undruggable” nuclear PD-L1 expression and impair cancer cell plasticity of metastatic TNBCs that currently have limited effective therapeutic options.

e13100 Background: Aurora-A mitotic kinase (AURKA) plays a pivotal role in Triple Negative Breast Cancer (TNBC) progression through activation of epithelial to mesenchymal transition (EMT)-mediated cancer cell plasticity. Cancer cells that undergo EMT acquire a CD44high/CD24low and/or ALDHhigh cancer stem-like phenotype (Cancer Stem Cells) characterized by high self-renewal capacity and intrinsic drug resistance. Cancer Stem Cells (CSCs) also shows high PD-L1 expression that plays a critical role in inducing tumor immune escape through CD8+ T-cells exhaustion. For these reasons, molecular characterization of oncogenic pathways that promote stemness and intra-tumoral PD-L1 expression is essential to evaluate whether novel combinatorial therapeutic strategies aimed to inhibit cancer cell plasticity, immune evasion capacity and organ metastasis may impact cancer progression. Methods: (1) TNBC Models: MDA-MB 231 and variant MDA-MB 231/LM cells, and unique TNBC-M40 cells established from metastatic PDXs; (2) In Vitro Studies: TNBC cells were treated with the AURKA inhibitor alisertib (50nM). PD-L1 expression was evaluated by immunoblotting assay. Apoptosis was measured by immunofluorescence using Cleaved-PARP antibodies. To assess stemness capacity, TNBC cells were cultured under non-adherent conditions to form mammospheres. ALDH activity (functional stemness marker) was measured using the Aldefluor Kit. (3) In Vivo Studies: 1x106 MDA-MB 231/LM cells were injected into the mammary fat pad of humanized NSG-CD34+ female mice. Animals were randomized into four groups (5 mice per group) and treated with anti-PD-L1 ICI atezolizumab (20 mg/Kg) and alisertib (25 mg/Kg) as monotherapy or in combination. Control groups were treated with saline solution placebo. PD-L1, CD44, Vimentin expression and tumor infiltration of CD8+ T-cells were assessed by immunofluorescence. Results: Treatment of MDA-MB 231/LM xenografts with alisertib decreased the levels of CD44 and PD-L1 expression and increased CD8+ T-cells tumor infiltration. Because PD-L1 genetic targeting enhanced in vitro alisertib-induced apoptosis, MDA-MB 231/LM xenografts were treated with alisertib and the anti-PD-L1 ICI atezolizumab. Combination of atezolizumab and alisertib resulted in the reduced expression of the EMT marker vimentin that was linked to lack of organ metastasis. Remarkably, combination of atezolizumab and alisertib also induced a significant reduction of ALDHHigh CSC in ex-vivo 3D-Organoids established from TNBC-M40 cells. Conclusions: This study provides innovative pre-clinical rationale for combining AURKA inhibitors with ICIs to impair cancer cell plasticity, immune evasion capacity and halt the progression of metastatic TNBCs that currently has limited effective therapeutic options.