Discovery of Kinesin KIF18A Inhibitor ATX020: Tactical Application of Silicon Atom Replacement.

Microsatellite instability (MSI) is an established marker of good prognosis in colorectal cancer (CRC). Chromosomal instability (CIN) is strongly negatively associated with MSI and has been shown to be a marker of poor prognosis in a small number of studies. However, a substantial group of "double-negative" (MSI-/CIN-) CRCs exists. The prognosis of these patients is unclear. Furthermore, MSI and CIN are each associated with specific molecular changes, such as mutations in KRAS and BRAF, that have been associated with prognosis. It is not known which of MSI, CIN, and the specific gene mutations are primary predictors of survival. We evaluated the prognostic value (disease-free survival, DFS) of CIN, MSI, mutations in KRAS, NRAS, BRAF, PIK3CA, FBXW7, and TP53, and chromosome 18q loss-of-heterozygosity (LOH) in 822 patients from the VICTOR trial of stage II/III CRC. We followed up promising associations in an Australian community-based cohort (N=375). In the VICTOR patients, no specific mutation was associated with DFS, but individually MSI and CIN showed significant associations after adjusting for stage, age, gender, tumor location, and therapy. A combined analysis of the VICTOR and community-based cohorts showed that MSI and CIN were independent predictors of DFS (for MSI, hazard ratio (HR)=0.58, 95% confidence interval (CI) 0.36-0.93, and P=0.021; for CIN, HR=1.54, 95% CI 1.14-2.08, and P=0.005), and joint CIN/MSI testing significantly improved the prognostic prediction of MSI alone (P=0.028). Higher levels of CIN were monotonically associated with progressively poorer DFS, and a semi-quantitative measure of CIN was a better predictor of outcome than a simple CIN+/- variable. All measures of CIN predicted DFS better than the recently described Watanabe LOH ratio. MSI and CIN are independent predictors of DFS for stage II/III CRC. Prognostic molecular tests for CRC relapse should currently use MSI and a quantitative measure of CIN rather than specific gene mutations.

The 2 chief pathways implicated in colorectal carcinogenesis, microsatellite instability and chromosomal instability, are not present in 20% to 37% of cases. To determine whether the CpG island methylator phenotype (CIMP) pathway, characterized by simultaneous methylation of several known tumor suppressor genes, is the principal underlying mechanism in cases without chromosomal or microsatellite instability, and to determine the significance of CIMP pathway and BRAF mutations in microsatellite-stable (MSS) cases. Clinicopathologic features and chromosomal instability status by loss of heterozygosity analysis were determined in 83 cases of colorectal cancer in which microsatellite instability, CIMP status, BRAF mutations, and KRAS mutations were previously known. Microsatellite instability was present in 14 cases (17%). Of the 69 MSS cases (83%), chromosomal instability manifested by LOH involving at least one locus was observed in 53 cases (64%). Hence, 16 (19%) of 83 colorectal cancer cases showed neither microsatellite instability nor chromosomal instability. These cases had a low incidence of CIMP (3/16; 19%) and BRAF mutation (1/16; 6%). The 5-year survival in these cases was significantly better compared with MSS cases with chromosomal instability (80% vs 54%, P = .02). BRAF mutations were identified in 10 MSS cases (15%). BRAF mutation in MSS cases correlated significantly with high-level chromosomal instability (P = .009) and poor 5-year survival (0% vs 70%, P < .001). CIMP does not appear to play a key role in colorectal cancer without microsatellite instability and chromosomal instability. These cases have a better survival, probably related to absence of significant chromosomal instability. BRAF mutations in MSS cases are associated with high levels of chromosomal instability that are likely responsible for the adverse outcomes in these cases.

Chromosome instability (CIN) generates genetic and karyotypic diversity that is common in hematological malignancies. Low to moderate levels of CIN are well tolerated and can promote cancer proliferation. However, high levels of CIN are lethal. Thus, CIN may serve both as a prognostic factor to predict clinical outcome and as a predictive biomarker. A retrospective study was performed to evaluate CIN in acute myeloid leukemia (AML). Chromosome mis-segregation frequency was correlated with clinical outcome in bone marrow core biopsy specimens from 17 AML cases. Additionally, we induced chromosome segregation errors in AML cell lines with AZ3146, an inhibitor of the Mps1 mitotic checkpoint kinase, to quantify the phenotypic effects of high CIN. We observed a broad distribution of chromosome mis-segregation frequency in AML bone marrow core specimens. High CIN correlated with complex karyotype in AML, as expected, although there was no clear survival effect. In addition to CIN, experimentally inducing chromosome segregation errors by Mps1 inhibition in AML cell lines causes DNA damage, micronuclei formation, and upregulation of interferon stimulated genes. High levels of CIN appear to be immunostimulatory, suggesting an opportunity to combine mitotic checkpoint inhibitors with immunotherapy in treatment of AML.

Decision letter: Interrogating the precancerous evolution of pathway dysfunction in lung squamous cell carcinoma using XTABLE

Editor's evaluation: Interrogating the precancerous evolution of pathway dysfunction in lung squamous cell carcinoma using XTABLE

Small cell lung cancer (SCLC), the most aggressive form of lung cancer, is notable for early dissemination and impressive, yet transient, responses to frontline chemotherapy that are rapidly undone by refractory relapses. To gain a better understanding of resistance mechanisms, we generated circulating tumor cell-derived xenograft (CDX) models from liquid biopsies of SCLC patients and treated them with chemotherapy or targeted agents. As expected, CDXs demonstrate similar chemotherapy response to the patient from whom they were derived. We have previously established that baseline chemosensitive CDXs are molecularly homogeneous at the single cell level, while chemoresistant CDXs exhibit increased intratumoral heterogeneity (ITH) with distinct variations in gene expression between cancer cell populations. We hypothesize that paired analyses of chemosensitive CDXs will demonstrate increased ITH after developing acquired resistance and are likely to offer more specific insights into resistance mechanisms than prior work with unrelated CDX models. Platinum-sensitive CDXs were treated with cisplatin or DNA damage response targeted therapies (PARP inhibitor or CHK inhibitor) continuously until tumor progression was observed. To identify transcriptional changes associated with onset of resistance, single-cell RNAseq analysis was performed on vehicle-treated and relapsed CDX tumors. We found globally increased ITH including heterogeneous expression of therapeutic targets and potential resistance pathways, such as EMT, between cellular subpopulations following treatment-resistance. Relapse was consistently associated with increased ITH score (P&amp;lt;0.001) and cell cluster number. To determine whether transcriptional diversity in either therapeutic targets (ex., MYC, DLL3, TOP2A, PARP1, CHEK1, EZH2, etc.) or EMT genes (ex., ZEB1, ZEB2, TWIST1, VIM, AXL) was detectable between the paired tumors after onset of resistance, we compared transcriptional differences between clusters present in vehicle-treated versus cisplatin or targeted therapy-treated tumors. We discovered the emergence of new cell clusters in relapsed tumors in all CDX models (4 CDXs, 3 distinct treatments), including clusters expressing EMT or NOTCH signaling genes. These data suggest that, in response to treatment, SCLC develops increasing transcriptional ITH marked by concurrent, diverse resistant cell clusters. Clinically, these data underscore the importance of maximizing and maintaining the initial response in platinum-sensitive SCLC tumors and highlight the intrinsic transcriptional fluidity underlying SCLC's profound treatment resistance following initial therapy. Citation Format: C. Allison Stewart, Carl M. Gay, Yuanxin Xi, Santhosh Sivajothi, Junya Fujimoto, Patrice M. Hartsfield, Hai Tran, Stephen G. Swisher, Jack A. Roth, Jianjun Zhang, Bonnie Glisson, John V. Heymach, Ignacio Wistuba, Paul Robson, Jing Wang, Lauren A. Byers. Paired, single-cell profiling of circulating tumor cell-derived xenograft models of small cell lung cancer reveals intratumoral heterogeneity and emergence of new cell clusters following treatment relapse [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 1496.

Telomere Dysfunction and DNA Damage Checkpoints in Diseases and Cancer of the Gastrointestinal Tract

Aneuploidy, a karyotype that differs from a multiple of the haploid, is a hallmark of cancer. Aneuploidy can be caused by an ongoing rate of chromosome missegregation during mitosis, known as whole chromosomal instability (CIN). Work from our lab and others has shown that while low rates of CIN can promote tumorigenesis, high levels of CIN are tumor suppressive. Combining two insults that each cause low CIN results in high CIN, cell death, and tumor suppression. We recently showed that the chemotherapy drug paclitaxel causes CIN due to multipolar divisions in breast cancer. Preliminary evidence suggests that tumors which exhibit CIN prior to therapy are sensitized to paclitaxel because it increases their level of CIN over a maximally tolerated threshold. Radiation also induces CIN, and rectal adenocarcinomas which exhibit CIN prior to treatment are sensitized to chemoradiation therapy. Human papillomavirus (HPV), specifically HPV subtype 16, is a growing cause of head and neck cancer worldwide. In cervical cancer, HPV is associated with CIN, which has been attributed largely to the expression of its E6 and E7 oncoproteins. HPV+ head and neck cancer patients show improved responses to radiation therapy than HPV- head and neck patients, potentially due to an increased basal rate of CIN. Here, we tested the ability of HPV E6, E7, or E6+E7 expression to induce CIN in normal oral keratinocytes (NOKs). Expression of E6 alone robustly induced misaligned chromosomes and multipolar spindles with supernumerary centrosomes, while E7 expression alone produced more moderate effects. Combined expression of E6 and E7 exacerbated the phenotypes observed in NOKs expressing E6 alone. Expression of E6, either alone or in combination with E7 caused reduced protein levels of the mitotic kinesin CENP-E, which is sufficient to induce misaligned chromosomes. Interestingly, HPV+ patient-derived xenograft (PDX) models of head and neck squamous cell carcinoma showed less evidence of mitotic defects than NOKs expressing E6 alone. A subset of HPV+ PDX tumors showed an increase in misaligned chromosomes when compared to HPV- tumors, but no increase in lagging chromosomes or multipolar spindles was observed. Together, these data suggest HPV infection causes an incompletely penetrant increase in specific types of CIN in head and neck cancer and that a basal rate of CIN may be predictive of treatment response independent of HPV status. Citation Format: Laura C. Funk, Denis L. Lee, Randall J. Kimple, Paul F. Lambert, Beth A. Weaver. HPV oncoproteins cause specific types of chromosomal instability in head and neck cancer [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 1846.

Sky-1214, a Small Molecule Splicing Modulator Targeting FANCL and Fanci, Provides a New Mechanism of Action Targeting Multiple Myeloma and Non-Hodgkin's Lymphoma

Brain metastasis in colorectal cancer (mCRC) poses a significant clinical challenge with poor outcomes. Our study, the largest spatial analysis reported to date, aimed to uncover unique cellular and genomic features facilitating metastatic lesion development. Employing spatial transcriptomics on 60 patients' surgical resections, we validated our findings with single-cell RNA sequencing (scRNA-seq), whole-genome sequencing (WGS), and ex vivo functional studies in tumor slice cultures. We obtained spatially distinct gene expression profiles of metastatic tumor cells, tumor microenvironment (TME) and brain parenchymal lineages. Our analysis identified spatially heterogenous upregulation of PI3K, MAPK, EGFR, p53, and TGFβ signaling in metastatic tumor cells. Tumor cells had high levels of chromosomal instability (CIN) marked by recurrent amplifications in chromosomes 6, 7, 17, and 20. WGS from three independent cohorts revealed significantly higher CIN in mCRC brain compared to primary CRC or mCRC from other organs. Spatial neighborhood analysis identified an immune desert phenotype with lack of proximity between lymphocytes and metastatic tumor cells. However, mCRC brain contained abundant plasma cells identifying a unique TME niche feature. Significant proximity between tumor cells and endothelial cells underscored their potential role in tumor seeding and growth. Spatially aware differential expression analysis demonstrated adaptive responses in the brain parenchyma. This included increased Golgi-ER processing and Rho GTPase signaling in neurons and astrocytes adjacent to metastatic tumor cells, respectively. Macrophages were characterized by high expression of pro-fibrogenic genes. Increased SPP1 ligand expression from these macrophages interacted with corresponding receptors on spatially proximal fibroblasts, identifying a key niche reorganization feature that can promote metastatic lesion growth. To evaluate the translational potential of our findings, we examined the effects of perturbating the metastatic niche using scRNA-seq. We established a tumor slice culture from a mCRC brain surgical resection, which maintained all original cell states. Despite elevated signaling pathway activity, metastatic tumor cells were resistant to regorafenib, a multi-receptor tyrosine kinase inhibitor. In contrast, pirfenidone, an anti-fibrotic and anti-inflammatory small molecule, successfully reduced extracellular matrix-associated gene expression programs in fibroblasts in the TME. Our analysis identified distinct properties of mCRC brain that can serve as therapeutic targets, including high levels of chromosomal instability, remodeling of brain parenchyma, lymphocyte evasion, plasma cell abundance and macrophage-fibroblast interactions. Citation Format: Anuja Sathe, Aparajita Khan, Ji In Kang, Rithika Meka, Susan M. Grimes, Andrew S. Luksik, Michael Lim, Claudia Petrisch, Christopher M. Jackson, Hannes Vogel, Melanie Gephart, Summer Han, Hanlee P. Ji. Spatial profiling of human colorectal cancer brain metastasis identifies chromosomal instability with adaptive niche cellular reorganization and reprograming [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1272.

Variable Levels of Chromosomal Instability and Mitotic Spindle Checkpoint Defects in Breast Cancer

KIF18A is a plus-end directed kinesin known to play a role in mitosis by facilitating chromosome alignment and spindle microtubule dynamics. Knockdown or knockout of KIF18A has been shown to inhibit proliferation in cells with whole genome doubling or aneuploidy, cellular states characterized by chromosomal rearrangements. These alterations render cells particularly vulnerable to disrupted mitosis upon KIF18A loss. As such, KIF18A is a compelling target in a subset of tumors with chromosomal instability (CIN), which have ongoing chromosomal segregation defects. We have identified potent and selective small molecule inhibitors of KIF18A. A proprietary tool compound, ATX-21020, inhibits KIF18A ATPase activity with an IC50 of 14.5 nM; KIF18A is selectively inhibited over other kinesins including CENPE (IC50 &amp;gt; 10 µM) and EG5 (IC50 5.87 µM). ATX-21020 exhibits robust cellular activity, with anti-proliferative activity observed in chromosomally instable ovarian cancer cell lines such as OVCAR-3 (IC50 53.3 nM) and OVCAR-8 (IC50 0.54 µM). Upon treatment with ATX-21020, Annexin V is robustly induced in sensitive cell lines, demonstrating that KIF18A-dependent cell lines undergo apoptosis upon KIF18A inhibition. In KIF18Ai sensitive cells, a dose-dependent upregulation of phospho-Histone H3 and gamma-H2AX is observed upon ATX-21020 treatment in OVCAR-3 and OVCAR-8 cells, consistent with the induction of mitotic arrest and DNA damage. In contrast, CIN negative Ovarian cancer cells that are insensitive to KIF18A loss, such as A2780 and OVK18, proliferate normally upon ATX-21020 treatment, and Annexin V, phospho-Histone H3, and gamma-H2AX are not induced. In sensitive cell lines, ATX-21020 induces a dose-dependent cell cycle arrest in G2/M, consistent with the role of KIF18A in facilitating mitosis. OVCAR-3 cells treated with ATX-21020 exhibit fragmented nuclei and malformed mitotic spindles, in contrast to A2780 cells where cell division is unaffected, suggesting that KIF18A is dispensable in CIN negative cells. This selective dependency is also observed in vivo, where administration of ATX-21020 leads to dose-dependent tumor growth inhibition in an OVCAR-3 cancer cell line-derived xenograft model, with significant tumor regression observed at 100 mpk/day. In contrast, OVK18 tumors are unaffected by ATX-21020 administration, as expected based on the lack of cell cycle and proliferation effects in that cell line. Together, these data demonstrate a critical role for KIF18A in facilitating mitosis in a subset of chromosomally instable cells. These effects are selective, with KIF18A-independent cell lines proceeding through the cell cycle normally in the presence of inhibitors. Small molecule inhibition of KIF18A is therefore expected to have robust efficacy in solid tumors with high levels of chromosomal instability such as Ovarian cancer. Citation Format: Maureen S. Lynes, Sanjoy Khan, Taylor Hotz, Brian A. Sparling, Mary-Margaret Zablocki, Deepali Gotur, P Ann Boriack-Sjodin, Kenneth W. Duncan, Stephen J. Blakemore, Serena J. Silver, Robert A. Copeland. Inhibition of KIF18A leads to mitotic arrest and robust anti-tumor activity in chromosomally instable tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3337.

KIF18A is a plus-end directed kinesin known to play a role in mitosis by facilitating chromosome alignment and spindle microtubule dynamics. Knockdown or knockout of KIF18A has been shown to inhibit proliferation in cells with whole genome doubling or aneuploidy, cellular states characterized by chromosomal rearrangements. These alterations render cells particularly vulnerable to disrupted mitosis upon KIF18A loss. As such, KIF18A is a compelling synthetic lethality target in a subset of tumors with chromosomal instability (CIN), which have ongoing chromosomal segregation defects. We have identified potent and selective small molecule inhibitors of KIF18A. A proprietary tool compound, ATX020, inhibits KIF18A ATPase activity with an IC50 of 0.014 µM; KIF18A is selectively inhibited over other kinesins including CENPE (IC50 &amp;gt; 10 µM) and EG5 (IC50 5.87 µM). ATX020 exhibits robust cellular activity, with anti-proliferative activity observed in chromosomally instable ovarian cancer cell lines such as OVCAR-3 (IC50 0.053 µM) and OVCAR-8 (IC50 0.54 µM). Upon treatment with ATX020, Annexin V is robustly induced in sensitive cell lines, demonstrating that KIF18A-dependent cell lines undergo apoptosis upon KIF18A inhibition. In KIF18Ai sensitive cells, a dose-dependent upregulation of phospho-Histone H3 and gamma-H2AX is observed upon ATX020 treatment in OVCAR-3 and OVCAR-8 cells, consistent with the induction of mitotic arrest and DNA damage. In these sensitive cell lines, ATX020 induces a dose-dependent cell cycle arrest in G2/M, consistent with the role of KIF18A in facilitating mitosis. OVCAR-3 cells treated with ATX020 exhibit fragmented nuclei and malformed mitotic spindles. In contrast, CIN negative Ovarian cancer cells that are insensitive to KIF18A loss, such as A2780 and OVK18, proliferate normally upon ATX020 treatment, cell division is unaffected, and no induction of Annexin V, phospho-Histone H3, and gamma-H2AX is observed. This selective dependency is also observed in vivo, where administration of ATX020 leads to dose-dependent tumor growth inhibition in an OVCAR-3 cancer cell line-derived xenograft model, with significant tumor regression observed at 100 mpk/day. In contrast, OVK18 tumors are unaffected by ATX020 administration, as expected based on the lack of cell cycle and proliferation effects in that cell line. Together, these data demonstrate a critical role for KIF18A in facilitating mitosis in a subset of chromosomally instable cells. These effects are selective, with KIF18A-independent cell lines proceeding through the cell cycle normally in the presence of inhibitors. Small molecule inhibition of KIF18A is therefore expected to have robust efficacy in solid tumors with high levels of chromosomal instability such as Ovarian cancer. Citation Format: Maureen S. Lynes, Laura Ghisolfi, Sanjoy Khan, Taylor Hotz, Brian A. Sparling, Mary-Margaret Zablocki, Deepali Gotur, P. Ann Boriack-Sjodin, Kenneth W. Duncan, Stephen J. Blakemore, Serena J. Silver, Robert A. Copeland. KIF18A inhibition, via ATX020, leads to mitotic arrest and robust anti-tumor activity through a synthetic lethal interaction with chromosome instability [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr PR001.

Small cell lung cancer (SCLC) accounts for 14% of lung cancer diagnoses in the United States and is characterized by rapid onset of chemoresistance and poor clinical outcomes. Once considered a homogeneous disease, recent analyses of SCLC have identified intratumoral heterogeneity (ITH) with respect to NOTCH signaling, ASCL1/NEUROD1 balance and MYC amplification - all of which are potential mechanisms underlying SCLC's aggressive and refractory biology. Unfortunately, patient-derived models of SCLC with which to better characterize the molecular profiles of refractory SCLC are scarce. To address this, we generated circulating tumor cell-derived xenograft (CDX) models from liquid biopsies of patients with treatment-naïve or relapsed SCLC. Each CDX model underwent pathological review to confirm tumors were consistent with SCLC based on histology and standard immunohistochemical markers (e.g., TTF1, chromogranin A, synaptophysin, NCAM). Sequencing of these models revealed mutations typical of SCLC (e.g. TP53, RB1), which were maintained in vivo over multiple passages. Importantly, each model's in vivo response to cisplatin matched the patient's platinum response at the time of CDX generation. At the proteomic level, platinum-resistant models exhibited mTOR activation, increased SOX2 and ATM, and reduced E-cadherin, suggesting a shift toward EMT and cancer stem cell expansion may contribute to resistance. To investigate ITH, we analyzed single-cell gene expression profiles by RNAseq using a droplet-based Chromium Single Cell system that analyzed a filtered subsample of 2000 cells per tumor. Consistent with SCLC, all CDX models contained large numbers of cells expressing neuroendocrine-specific genes (SYP, CHGA). However, Principle Component Analysis revealed that cells from chemosensitive CDX models had distinct expression profiles from resistant models. Using our published EMT gene signature, we found that resistant models had higher proportions of mesenchymal (vs. epithelial) cells. Several other distinctions between sensitive and resistant models were detected at the single-cell level but not in bulk RNA and protein analyses, suggesting that single-cell resolution can identify occult platinum-resistant subpopulations. For example, higher proportions of ASCL1- and DLL3-expressing cells were associated with platinum sensitivity, whereas a shift toward predominant NEUROD1-expression was observed with resistance. Cells expressing each of these three genes were identified across all tumors, suggesting platinum-sensitive and resistant subpopulations are ubiquitous but that even subtle shifts in the fractional distribution of these subsets can exert significant impact on response. These data support further use of single-cell analysis to explore the role of ITH as a driver of drug resistance in SCLC. Citation Format: C. Allison Stewart, Carl M. Gay, Yuanxin Xi, Siva V., Junya Fujimoto, Pan Tong, Lixia Diao, Lerong Li, Mohan Bolisetty, Neda Kalhor, Patrice Lawson, Mayra Vasquez, Hai Tran, Ignacio I. Wistuba, Bonnie Glisson, Jianjun Zhang, Stephen G. Swisher, Jack A. Roth, John V. Heymach, Paul Robson, Jing Wang, Lauren A. Byers. Single-cell profiling of small cell lung cancer circulating tumor cell-derived xenograft models reveals intratumoral heterogeneity among mediators of chemoresistance [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 990.

Taxanes remain among the most effective agents for the treatment of breast cancer. However, ˜50% of patients who receive this therapy do not have a clinical response. Previously, we identified the mechanistic effects of taxane therapy by sampling primary human breast tumors 20 hours after infusion (Sci Trans Med 26:229ra43, 2014). We determined that paclitaxel caused cells to transit mitosis on multipolar spindles, resulting in increased chromosome gains and losses. Preclinical models suggest that tumors have enhanced sensitivity if they have baseline chromosome gains/losses, known as chromosome instability (CIN). The aim of this study was to compare CIN against other predictive biomarkers in a retrospective cohort of advanced breast cancer patients who receive single-agent taxane therapy. Methods: We identified 36 subjects with metastatic breast cancer and measurable disease who received taxane therapy and had available archived tissue. Responses were determined by RECIST 1.1 criteria. We evaluated chromosome number on a cell-by-cell basis using 6 centromeric FISH probes. Additionally we measured rates of proliferation by phospho-histone H3 (p-HH3) and Ki67, previously reported biomarkers, β-tubulin III, P-gp1 and MAD1 by quantitative immunofluorescence. CIN was estimated as the fraction of cells with non-modal chromosome numbers across chromosomes 3, 4, 7, 9, 10, and 17. Results: Of the 36 subjects, 19 had ER/PR+HER2- disease (53%), 9 had HER2-positive disease (25%), and 8 TNBC (22%). Single-agent chemotherapy was used for all including paclitaxel in 16, nab-paclitaxel in 17, and docetaxel in 3 (HER2+ patients received concurrent trastuzumab). RECIST responses ranged from disease progression (8%, n=3), stable disease (50%, n=18), and partial response (42%, n=15). No complete responses were observed. Time on therapy ranged from 1.4 months to 28 months. No statistically significant correlations were found between tumor type or prior chemotherapy and response to taxanes. Archived metastatic samples were available for 21 subjects. Analysis showed large variations in Ki67, pHH3, β-tubulin III, P-gp1, MAD1, and CIN amongst samples. The strongest correlation was found between tumor response and high levels of CIN, with a Spearman's correlation coefficient of 0.38 (p=0.04). Surprisingly, there was an inverse correlation between Ki67 and taxane response, although this did not reach statistical significance. Biomarkers correlated with response to taxaneN=21Ki67 (%)pHH3 (H)βtub3 (H)P-gp1 (H)MAD1 (H)CIN (%)min0.850.000.673.002.8324.2max45.14.8915129923165.8Spearman rho-0.3650.242-0.1650.232-0.08960.382p (1-tailed)0.05190.1400.2370.1550.3500.0425H = H-score; CIN is estimated as % of cells with non-modal chromosomes Conclusions: Chromosomal instability is a promising biomarker for predicting sensitivity to taxane therapies. Additional studies will be necessary to validate CIN as a biomarker and to determine whether 6-chromosome FISH can be supplanted by low-pass single-cell DNA sequencing. Citation Format: Esbona K, Jin N, Correia-Staudt KL, Lager AM, Heidke T, Laffin J, Weaver BA, Burkard ME. Chromosomal instability predicts taxane sensitivity in breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P4-03-09.