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Multinucleated H2B-RFP Tub-GFP MDA-MB-231 Multipolar Division

Mononucleated H2B-RFP Tub-GFP MDA-MB-231 Bipolar Division

Purpose:As monotherapies, eribulin (chemotherapy) and pembrolizumab (immunotherapy) have shown promise for patients with metastatic triple-negative breast cancer (mTNBC). This phase Ib/II study examined eribulin plus pembrolizumab as a potential mTNBC treatment in first-line and later-line settings.Patients and Methods:In this open-label, single-arm, phase Ib/II study, eligible patients had mTNBC, measurable disease, and ≤2 prior systemic anticancer therapies in the metastatic setting. Patients were enrolled by number of prior systemic anticancer therapies (stratum 1: 0 vs stratum 2: 1–2) in the metastatic setting and further analyzed by tumor programmed death-ligand 1 (PD-L1) expression status. All patients received intravenous eribulin 1.4 mg/m2 on day 1 and day 8, plus intravenous pembrolizumab 200 mg on day 1, of 21-day cycles. The primary objectives were the safety, tolerability, and objective response rate (ORR) of this combination.Results:The study included 167 patients (phase Ib, n = 7; phase II, n = 160). The most common treatment-emergent adverse events were fatigue (66%), nausea (58%), peripheral sensory neuropathy (41%), alopecia (40%), and constipation (37%). ORRs were 25.8% [95% confidence interval (CI): 15.8–38.0] for stratum 1 (n = 66) and 21.8% (95% CI: 14.2–31.1) for stratum 2 (n = 101). Patients with PD-L1–positive tumors (combined positive score ≥1) had numerically higher ORR than those with PD-L1–negative tumors, particularly in stratum 1 [stratum 1: 34.5% (95% CI: 17.9–54.3) vs 16.1% (95% CI: 5.5–33.7); stratum 2, 24.4% (95% CI: 12.9–39.5) vs 18.2% (95% CI: 8.2–32.7)].Conclusions:Eribulin plus pembrolizumab was generally well tolerated and showed promising antitumor activity in mTNBC. Efficacy outcomes appeared influenced by line of therapy and PD-L1 status.

Supplementary Information

Triple-negative breast cancer (TNBC) commonly develops resistance to chemotherapy, yet markers predictive of chemoresistance in this disease are lacking. Here, we define WNT10B-dependent biomarkers for β-CATENIN/HMGA2/EZH2 signaling predictive of reduced relapse-free survival. Concordant expression of HMGA2 and EZH2 proteins is observed in MMTV-Wnt10bLacZ transgenic mice during metastasis, and Hmga2 haploinsufficiency decreased EZH2 protein expression, repressing lung metastasis. A novel autoregulatory loop interdependent on HMGA2 and EZH2 expression is essential for β-CATENIN/TCF-4/LEF-1 transcription. Mechanistically, both HMGA2 and EZH2 displaced Groucho/TLE1 from TCF-4 and served as gatekeepers for K49 acetylation on β-CATENIN, which is essential for transcription. In addition, we discovered that HMGA2-EZH2 interacts with the PRC2 complex. Absence of HMGA2 or EZH2 expression or chemical inhibition of Wnt signaling in a chemoresistant patient-derived xenograft (PDX) model of TNBC abolished visceral metastasis, repressing AXIN2, MYC, EZH2, and HMGA2 expression in vivo. Combinatorial therapy of a WNT inhibitor with doxorubicin synergistically activated apoptosis in vitro, resensitized PDX-derived cells to doxorubicin, and repressed lung metastasis in vivo. We propose that targeting the WNT10B biomarker network will provide improved outcomes for TNBC.Significance:These findings reveal targeting the WNT signaling pathway as a potential therapeutic strategy in triple-negative breast cancer.

Supplementary Section: 1) Seven supplementary figures and legends: A. Supplementary figure S1: TCGA analysis of WNT10B network. B. Supplementary figure S2: Human IHC analysis of WNT10B, HMGA2 and EZH2. C. Supplementary figure S3: Mouse IHC analysis of HMGA2 and EZH2. D. Supplementary figure S4: ChIP, qPCR and IB analysis on WNT10B/�CATENIN direct target genes. E. Supplementary figure S5: Characterization of CRISPR HMGA2KO and EZH2KO cells both in vitro and in vivo. F. Supplementary figure S6: Validation of WNT10B network on TNBC PDX tumors. G. Supplementary figure S7: Isobologram curves of ICG-001 and Doxorubicin activity. 2) Supplementary Material & Methods 3) Supplementary Table 1 (ST1) qPCR primers 4) Supplementary Table 2 (ST2) ChIP primers 5) 6) Supplementary Table 3 (ST3) drug combinatorial index

Supplementary Figure Legends 1-2 from Bidirectional Crosstalk between Leptin and Insulin-like Growth Factor-I Signaling Promotes Invasion and Migration of Breast Cancer Cells via Transactivation of Epidermal Growth Factor Receptor

Supplementary Figure 1 from Bidirectional Crosstalk between Leptin and Insulin-like Growth Factor-I Signaling Promotes Invasion and Migration of Breast Cancer Cells via Transactivation of Epidermal Growth Factor Receptor

Fig. S1. Supplementary data for large scale genomic aberrations illustrated in Figure 2, including: A) the distribution of mapped read density of WGS; B) PCR confirmation of translocation and fusion junctions of a superamplicon; and C) PCR and RT-PCR amplification of MGAM, ZFAND3, and the ZFAND3-MGAM fusion gene. Fig. S2. The distribution of mapped read density of WES, supplementary data for Figure 3. Fig. S3. Differentially expressed genes and functions among complex, simple carcinomas and normal mammary glands, supplementary data for Figure 3. Fig. S4. IHC images of histone modifications in canine normal mammary glands at 400X magnification, supplementary data for Figure 4. Fig. S5. IHC images of histone modifications in simple carcinomas, complex carcinomas and normal mammary glands at 200X magnification, supplementary data for Figure 5. Fig. S6. Supplementary data for homology between canine MCs and human breast cancers described in Figure 6. These include differentially expressed genes between ER+ and ER- carcinomas at FDR ≤ 0.1 and an example of PAM50 clustering indicating canine and human tumor IDs.

Supplementary Figure 2 from Bidirectional Crosstalk between Leptin and Insulin-like Growth Factor-I Signaling Promotes Invasion and Migration of Breast Cancer Cells via Transactivation of Epidermal Growth Factor Receptor

Background: Phosphatidylinositol-3-kinase (PI3K) activating mutations are found in 30-40% of breast cancers, and the PI3K inhibitor alpelisib is FDA-approved for PIK3CA-mutated hormone-receptor positive metastatic breast cancer (MBC). However, rates of intrinsic alpelisib resistance are as high as 40%, and there is an ongoing need for novel biomarkers to help guide patient selection. Circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs) may better reflect the heterogeneity of metastatic disease than tissue biopsy, and are amenable to longitudinal analysis. By combining targeted ctDNA sequencing with CTC transcriptional profiling and quantitation of PI3K pathway phospho-proteins, we have developed the first multiplex assay to simultaneously assess genomic alterations and CTC PI3K pathway activity via liquid biopsy. Methods: Peripheral blood was collected from MBC patients with known PIK3CA mutation status serially prior to starting new standard therapies and on treatment. Cell-free DNA was extracted from plasma and sequenced using a custom capture panel (IDT). CTCs were isolated with VERSA microfluidic technology integrating capture with downstream analysis. CTCs were captured immunomagnetically followed by RNA isolation on chip and RNA-seq, or staining on chip for phospho-AKT pS473 and total AKT or phospho-rpS6 pS235/S236 and total rpS6, followed by quantification of single cell phospho/total protein mean fluorescence ratios. Results: In a pilot cohort of 14 patients, mean CTC phospho-AKT/total AKT protein expression ratio was higher in patients with PIK3CA mutations compared to patients without mutations (0.37 vs 0.26, p Conclusion: We have demonstrated the feasibility of a comprehensive liquid biopsy for simultaneous monitoring of PI3K pathway mutations in ctDNA and PI3K pathway signaling in CTCs via transcriptional profiling and phospho-protein expression in patients with MBC. Future work will prospectively evaluate this assay in patients receiving alpelisib for PIK3CA-mutated MBC. This has the potential to complement PIK3CA mutation status as a biomarker of sensitivity to PI3K therapies, and may also provide a pharmacodynamic assessment of PI3K inhibitor activity in CTCs on treatment. Citation Format: Marina N. Sharifi, Kyle T. Helzer, Jamie M. Sperger, Matthew L. Bootsma, Hannah Krause, Cole S. Gilsdorf, Serena K. Wolfe, Zachary Kauffman, Amye J. Tevaarwerk, Mark E. Burkard, Amanda M. Parkes, Ruth M. O'Regan, Kari B. Wisinski, Shuang G. Zhao, Joshua M. Lang. Simultaneous longitudinal assessment of PIK3CA genomic mutations and PI3K pathway activity in circulating tumor cells in metastatic 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 1955.