Abstract Background: TTK (Threonine Tyrosine Kinase also known as Monopolar spindle 1), is a dual-specificity serine-threonine kinase critical for anaphase promoting complex inhibition at the spindle assembly checkpoint and is required for chromosome alignment and error correction. TTK inhibition results in premature mitosis exiting with unattached chromosomes potentially leading to aneuploidy and cell death. High TTK tumor levels correlate with worse prognosis and contribute to the survival and proliferation of aneuploid cells. CFI-402257, a potent and selective inhibitor of TTK, inhibits the growth of a variety of human cancer-derived cell lines. A first-in-human phase 1 study of CFI-402257 (NCT02792465) demonstrated a tolerable safety profile when enrolled as a monotherapy in solid tumors, and in combination with fulvestrant in hormone receptor positive, HER2 negative (HR+/HER2-) breast cancer1. The dose for expansion was 168 mg, dose limiting toxicity was dose-dependent neutropenia which was manageable and reversible. Investigator-confirmed partial responses (cPR) were observed in 5 pts (8%) with 32 (50.8%) exhibiting disease control. In the HR+/HER2- breast cancer population previously treated with cyclin dependent kinase 4/6 inhibitors (CDK4/6i) and aromatase inhibitors (N=25), there were 4 cPR’s with a median duration of response of 223 days, with responses emerging after 2 cycles of therapy. Responses were observed with CFI-402257 as a single agent and in combination with fulvestrant. Based on these data, study TWT-203 will focus on advanced solid tumors for dose confirmation then focus on advanced HR+/HER2- breast cancers in combination with an approved endocrine therapy. Methods: In TWT-203 study, safety and clinical activity of CFI-402257 monotherapy will be evaluated in patients (pts) with advanced solid tumors (Part A) or in combination with fulvestrant in pts with HR+/HER2- advanced breast cancer (Part B). Part A will confirm the RP2D using a 3+3 design with a starting dose of 126 mg daily. Part B evaluates CFI-402257 in combination with fulvestrant in pts with HR+/HER2- advanced breast cancer following progression on prior CDK4/6i and endocrine therapy. Efficacy endpoints include overall response rate and disease control rate. Safety endpoints include incidence of treatment emergent adverse events. Exploratory objectives include characterization of protein and molecular alterations relevant to the cell cycle and CFI-402257 response. Results: At data cutoff of 11 May 2023, 14 pts were enrolled. All received monotherapy treatment. Median treatment duration was 2.1 months (range, 0.2-7.3+). Median age was 67 years (57-76). Median number of prior regimens was 3.5 (2-12). Tumor types enrolled were colorectal (n=6, 43%), breast (3, 21%), and endometrial, hepatocellular, leiomyosarcoma, pancreatic, and sarcoma (1 each, 7%). 4 dose levels, from 126 to 252 mg, were studied. Most pts (12, 86%) experienced ≥1 treatment emergent adverse event (TEAE). More than half of pts (8, 57%) experienced ≥1 treatment related TEAE. Most common TEAEs were fatigue (5, 36%) and nausea (4, 29%). 5 pts (36%) experienced TEAEs grade ≥3, most common were fatigue, neutrophil count decrease, white blood cell count decrease (2 pts each, 14.3%). 1 pt (7%) experienced a serious adverse event (hematuria), not related to study therapy. No patients discontinued treatment due to TEAE. All pts who ended treatment were due to disease progression (8, 57%). No grade 5 TEAEs occurred. No dose limiting toxicities were reported. Disease control rate (CR, PR, or SD ≥ 6 weeks from baseline) was 54.5% (6 pts/11): all achieved SD. 3 of the 6 pts remain on study. Conclusion: CFI-402257 is a potent inhibitor of TTK. It is well tolerated with manageable TEAEs, no dose limiting or treatment limiting toxicities, and no treatment related deaths. Dose expansion in the patient population of interest will commence. 1. John Hilton et al. Cancer Res 2023;83(5 Suppl): P6-10-13. Citation Format: Justin Call, Muralidhar Beeram, Alexander I. Spira, Mark Bray, Dih-Yih Chen, Long Kwei, Emily Roberts-Thomson, Trisha Denny, Roger Sidhu, Robert Wesolowski. TWT-203: PHASE 1b/2 STUDY OF CFI-402257 AS MONOTHERAPY IN ADVANCED SOLID TUMORS AND IN COMBINATION WITH FULVESTRANT IN PATIENTS WITH ER+/HER2- ADVANCED BREAST CANCER AFTER PROGRESSION ON PRIOR CDK4/6 INHIBITORS AND ENDOCRINE THERAPY [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-04-13.

Background: TTK (Threonine Tyrosine Kinase also known as Monopolar spindle 1), is a dual-specificity serine-threonine kinase critical for anaphase promoting complex inhibition at the spindle assembly checkpoint and is required for chromosome alignment and error correction. TTK inhibition results in premature mitosis exiting with unattached chromosomes potentially leading to aneuploidy and cell death. High TTK tumor levels correlate with worse prognosis and contribute to the survival and proliferation of aneuploid cells. CFI-402257, a potent and selective inhibitor of TTK, inhibits the growth of a variety of human cancer-derived cell lines. A first-in-human phase 1 study of CFI-402257 (NCT02792465) demonstrated a tolerable safety profile when enrolled as a monotherapy in solid tumors, and in combination with fulvestrant in hormone receptor positive, HER2 negative (HR+/HER2-) breast cancer1. The dose for expansion was 168 mg, dose limiting toxicity was dose-dependent neutropenia which was manageable and reversible. Investigator-confirmed partial responses (cPR) were observed in 5 pts (8%) with 32 (50.8%) exhibiting disease control. In the HR+/HER2- breast cancer population previously treated with cyclin dependent kinase 4/6 inhibitors (CDK4/6i) and aromatase inhibitors (N=25), there were 4 cPR’s with a median duration of response of 223 days, with responses emerging after 2 cycles of therapy. Responses were observed with CFI-402257 as a single agent and in combination with fulvestrant. Based on these data, study TWT-203 will focus on advanced solid tumors for dose confirmation then focus on advanced HR+/HER2- breast cancers in combination with an approved endocrine therapy. Methods: In TWT-203 study, safety and clinical activity of CFI-402257 monotherapy will be evaluated in patients (pts) with advanced solid tumors (Part A) or in combination with fulvestrant in pts with HR+/HER2- advanced breast cancer (Part B). Part A will confirm the RP2D using a 3+3 design with a starting dose of 126 mg daily. Part B evaluates CFI-402257 in combination with fulvestrant in pts with HR+/HER2- advanced breast cancer following progression on prior CDK4/6i and endocrine therapy. Efficacy endpoints include overall response rate and disease control rate. Safety endpoints include incidence of treatment emergent adverse events. Exploratory objectives include characterization of protein and molecular alterations relevant to the cell cycle and CFI-402257 response. Results: At data cutoff of 11 May 2023, 14 pts were enrolled. All received monotherapy treatment. Median treatment duration was 2.1 months (range, 0.2-7.3+). Median age was 67 years (57-76). Median number of prior regimens was 3.5 (2-12). Tumor types enrolled were colorectal (n=6, 43%), breast (3, 21%), and endometrial, hepatocellular, leiomyosarcoma, pancreatic, and sarcoma (1 each, 7%). 4 dose levels, from 126 to 252 mg, were studied. Most pts (12, 86%) experienced ≥1 treatment emergent adverse event (TEAE). More than half of pts (8, 57%) experienced ≥1 treatment related TEAE. Most common TEAEs were fatigue (5, 36%) and nausea (4, 29%). 5 pts (36%) experienced TEAEs grade ≥3, most common were fatigue, neutrophil count decrease, white blood cell count decrease (2 pts each, 14.3%). 1 pt (7%) experienced a serious adverse event (hematuria), not related to study therapy. No patients discontinued treatment due to TEAE. All pts who ended treatment were due to disease progression (8, 57%). No grade 5 TEAEs occurred. No dose limiting toxicities were reported. Disease control rate (CR, PR, or SD ≥ 6 weeks from baseline) was 54.5% (6 pts/11): all achieved SD. 3 of the 6 pts remain on study. Conclusion: CFI-402257 is a potent inhibitor of TTK. It is well tolerated with manageable TEAEs, no dose limiting or treatment limiting toxicities, and no treatment related deaths. Dose expansion in the patient population of interest will commence. 1. John Hilton et al. Cancer Res 2023;83(5 Suppl): P6-10-13. Citation Format: Justin Call, Muralidhar Beeram, Alexander I. Spira, Mark Bray, Dih-Yih Chen, Long Kwei, Emily Roberts-Thomson, Trisha Denny, Roger Sidhu, Robert Wesolowski. TWT-203: PHASE 1b/2 STUDY OF CFI-402257 AS MONOTHERAPY IN ADVANCED SOLID TUMORS AND IN COMBINATION WITH FULVESTRANT IN PATIENTS WITH ER+/HER2- ADVANCED BREAST CANCER AFTER PROGRESSION ON PRIOR CDK4/6 INHIBITORS AND ENDOCRINE THERAPY [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-04-13.

Background: CFI-400945 is a selective oral inhibitor of Polo-like Kinase 4 (PLK4), a controller of centriole duplication and mitotic progression identified by functional screening of genomically unstable breast cancer (BC). IND.237 (NCT01954316) is an open label, multicentre, phase 2 study in HER2 negative metastatic breast cancer (MBC) with 3 cohorts, 1 enriched for PTEN loss of function. Enrollment started in 2018 at 64mg based on a previously established recommended phase 2 dose (RP2D). The initial patients had higher than expected grade 3/4 neutropenia which led to a voluntary hold and dose de-escalation; the new RP2D was declared at 32mg as previously reported Here we report the results of the phase 2 study of CFI-400945 in advanced BC patients. Materials and Methods: 49 patients were enrolled across 3 cohorts: 1: triple negative; 2: ER+/HER2- PTEN low (by IHC); 3: ER+/HER2-, PTEN intact. The primary outcome is objective response rate (ORR); secondary outcomes included disease control rate (DCR) >16w, and safety. A Simon 2-stage design was used (9 – 25 pts planned for each cohort). CFI-400945 would be considered active if ≥3 responses were observed in any given cohort. Eligibility included ECOG 0-1, adequate organ function and receipt of at ≥1 prior line of cytotoxic chemotherapy in any setting including anthracycline taxane (unless contraindicated). Treatment was 32mg 7d on 7d off in cycle 1 (cycle length=28d), then continuously starting cycle 2. Safety assessments were performed each cycle and response (RECIST 1.1) every 2 cycles. Results: 60 patients have been screened, 49 enrolled: 10 were in initial dose ranging and were excluded from phase 2 response assessment. 10 patients were enrolled in cohort 1, 4 in cohort 2, and 25 in cohort 3. Table 1 presents patient characteristics and response results. 1 patient in cohort 3 has not had disease re-assessed at time of abstract submission. The most common adverse events have been cytopenias, nausea, fatigue, headache, constipation and vomiting. Less than 5% of patients experienced a non-hematologic AE > grade 3; 33% experienced ≥ grade 3 neutropenia. Conclusions: CFI-400945 32mg is well tolerated in this MBC population with moderate incidence of uncomplicated neutropenia. The TNBC cohort so this arm has been closed to further accrual for lack of responses. The PTEN loss group has been slow to accrue and remains open. Responses in the ER+/HER2- arm are encouraging – results from patients remaining on study are awaited and correlative studies to identify features associated with responses are underway. Acknowledgements: Sponsored by the Canadian Cancer Trials Group. Supported by Stand Up To Cancer Canada (scientific partner AACR) Canadian Cancer Society (CCS) Breast Cancer Dream Team Research Funding, Ontario Institute for Cancer Research (funding provided by the Government of Ontario) and grants from CCS to CCTG. Table 1. Table 1. Patient characteristics and response rates in each cohort treated with CFI-400945. Citation Format: David W. Cescon, John Hilton, Philippe Bedard, Phillip Blanchette, Rossanna C. Pezo, Ayesha Bashir, Vikaash Kumar, Terry L. Ng, Arif Awan, Anthony Lott, Jacques Antoun Raphael, Linda Hagerman, Mark Bray, Lindsay Muyot, Jesus Fuentes Antras, Lesley Seymour, Dongsheng Tu, Pierre-Olivier Gaudreau, Moira Rushton. A phase II study of CFI-400945 in patients with advanced/metastatic cancer: Canadian Cancer Trials Group (CCTG) IND.237 [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-07-14.

Background: TTK (also known as MPS1), a dual-specificity serine-threonine kinase, is critical for the spindle assembly checkpoint, chromosome alignment, and error correction in mitosis. Inhibition of TTK causes premature mitotic exit with unattached chromosomes, to result in chromosomal missegregation, aneuploidy, and cell death. CFI-402257 is a potent and highly selective inhibitor of TTK. Robust suppression of tumor growth was achieved upon oral dosing of single agent CFI-402257 in ER+/HER2- cell line and patient derived xenograft models. CFI-402257 demonstrated enhanced cytotoxicity in CDK4/6 inhibitor resistant ER+ breast cancer cell line models compared to parental cell lines, including those with RB1 loss. CFI-402257 has previously exhibited monotherapy and combination efficacy with a tolerable safety profile in ER+/Her2- Breast cancer patients in an ongoing clinical study which is updated here. Methods: This is an ongoing phase I, multi-center, dose escalation study (3+3 design) to determine the safety, tolerability, and maximum tolerated dose of CFI-402257 and to evaluate anti-tumor activity at the recommended phase 2 dose (RP2D). CFI-402257 was dosed once daily on a continuous schedule in 28-day cycles at a starting dose of 5 mg. Dose escalation included patients (pts) with advanced solid tumors. Dose expansion at the RP2D included pts with advanced solid tumors (Cohort A), advanced Her2-negative (ER+ or TNBC) with 1-4 prior lines of chemotherapy for metastatic disease (Cohort B), and ER+/Her2- breast cancer in combination with Fulvestrant (500 mg IM Day 1, 15 and 29 and then every 28 days) who have had prior treatment with an aromatase inhibitor in combination with a CDK4/6 inhibitor (≥ 3 months) and ≤1 prior chemotherapy for metastatic disease (Cohort C). Results: At data cutoff of May 2, 2022, 87 pts were enrolled. 86 pts (66 pts receiving monotherapy and 20 patients receiving combination therapy received ≥1 dose of study therapy and were analyzed for safety. One pt was not dosed due to elevated liver enzymes prior to first dose. The median age for mono was 61 years (range, 35-81) and for combo was 54 (range, 31-70). The median number of prior regimens mono and combo was 5 (range, 0-17; and 1-9, respectively). Tumor types in mono were breast cancer (27 pts), ovarian cancer (7 pts), GI cancer (3 pts), pancreatic cancer (3 pts), and other (30 pts). 20 breast cancer pts were enrolled in the combo. To date, 11 dose levels have been studied (range: 5 to 294 mg) in mono. There were 4 dose limiting toxicities (neutropenia >7days at 168mg/day, febrile neutropenia at 210mg/day, and neutropenia and colitis at 294mg/day, all grade 3). The RP2D of 168 mg was established. 3 pts in mono (biliary obstruction, febrile neutropenia, and pancytopenia) and 0 pts in combo discontinued study due to adverse events (AEs).Treatment emergent adverse events (TEAE’s) occurring in ≥30% of pts were fatigue (31 pts; 47%), nausea (30 pts; 46%), decreased appetite (22 pts; 33%), and diarrhea (21 pts; 32%) in mono; and nausea (11 pts; 55%) and diarrhea (7 pts; 35%) in combo. 35% of mono and 39% of combo AEs were considered related to CFI-402257 by the investigators. Grade ≥3 AEs and serious AEs were reported in 25 pts (38%) and 17 pts (26%), respectively in mono; and 5 pts (25%) and 3 pts (15%) in combo. The investigator assessed best overall response rate (partial response [PR] or better within the efficacy population) of 6% (PR; hepatocellular carcinoma n=1, breast cancer n=2 from n=47) in mono and 18% (PR; n=2 breast cancer) in combo (n=11), with additional patients still to be assessed. Conclusion: CFI-402257 is well tolerated as mono and combination with fulvestrant. Efficacy signals are emerging with pts in the combo cohort demonstrating anti-tumor activity. Additional efficacy will be updated at the time of the presentation. Citation Format: John Hilton, Daniel Renouf, David W. Cescon, Aaron Hansen, Alibiruni Abdul Razak, Lee-Anne Stayner, Trisha A. Denny, Emily Roberts-Thomson, Dih-Yih Chen, Mark Bray, Philippe Bedard. An update to a Phase I trial of CFI-402257, an oral TTK inhibitor, in patients with advanced solid tumors with HER2-negative breast cancer expansion cohorts [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-10-13.

CCTG IND.236: A Phase 1b trial of combined CFI-402257 and weekly paclitaxel in patients with HER2-negative (HER2-) advanced breast cancer (aBC) Philippe L. Bedard, Mihaela Mates, John Hilton, Nathalie Levasseur, Arif Awan, Amirrtha Srikanthan, David Cescon, Karen Gelmon, Andrew Robinson, Nancy Drummond-Ivars, Irene Li, Laleh Rastgou, Jackie Edwards, Linda Hagerman, Siwei Zhang, Mark Bray, Lesley Seymour, Moira Rushton-Marovac, Pierre-Olivier Gaudreau Background: CFI-402257 is a selective oral inhibitor of TTK protein kinase, a critical regulator of the mitotic spindle assembly checkpoint overexpressed in breast cancer (BC). CFI-402257 monotherapy has anti-proliferative and cytotoxic activity and enhances antitumor activity of paclitaxel in BC xenograft models. Material and methods: Primary objectives were to establish safety and Recommended Phase 2 dose (RP2D) of CFI-402257 combined with weekly paclitaxel (Phase 1b) and Overall Response Rate (ORR) as per RECIST 1.1 (Phase 2). Patients with HER2- aBC with adequate organ function, PS 0-1, previously treated with >1 non-taxane chemotherapy, were eligible. A 3+3 design was used for Phase 1b, with dose limiting toxicities (DLTs) assessed during cycle 1 (28 days). Starting dose CFI-402257 was 84mg (DL1 = 84mg, DL2 = 112mg, DL3 = 168mg, DL4 = 210mg and DL5 = 252mg) on a 2-day on, 5-day off schedule with paclitaxel 80mg/m2 day 1, 8, 15. Safety assessments were performed weekly (CTCAE v5.0) and response every 2 cycles. A Simon 2-stage design was used for Phase 2 (stage 2 required ≥4 responses in 17 evaluable patients from stage 1). Results: 37 patients received a total of 260 cycles including all 5 dose levels. Median age was 59; 92% ER+/HER2-; 49% PS1; 22% 3 prior chemotherapy lines; 41% 4 sites of metastatic disease, and 81% had received prior CDK4/6 inhibitors. Grade 3 hematological adverse events (AEs, all dose levels) were neutropenia (70%), lymphopenia (41%) and anemia (14%). Six DLTs occurred: 5 dose-related grade 4 neutropenia and 1 febrile neutropenia. Three DLTs occurred at DL3, two at DL4, and one at DL5. Three serious AEs (two at DL3, and one at DL4) at least possibly related to treatment were seen: 2 febrile neutropenia and 1 skin infection (all grade 3). Frequent AEs (˃5%; all dose levels) considered at least possibly related to treatment were: diarrhea (38%), nausea (30%), fatigue (27%), vomiting (16%), anorexia (14%), maculo-papular rash (14%), oral mucositis (11%), alopecia (11%) and pruritus (8%). DL3 (168mg) was selected as RP2D. ORR was 3/36=8% and 1/17=5.9% in all vs Phase 2 evaluable patients, respectively. Clinical Benefit Rate (CBR; defined as complete response, partial response or stable disease ˃16 weeks in duration) was 18/33=54.6% and 10/17=58.8% in all vs Phase 2 evaluable patients, respectively. During Phase 2, the 17 evaluable patients from stage 1 did not meet pre-specified threshold for anti-tumor activity to proceed to stage 2. Conclusions: CFI-402257 and paclitaxel was well tolerated, with neutropenia as the main toxicity. DL3 (168mg) was selected as RP2D. Phase 2 ORR and CBR was 5.9% and 58.8%, respectively; during Phase 2, the 17 evaluable patients from stage 1 did not meet the pre-specified threshold for anti-tumor activity to proceed to stage 2 and the trial was closed to accrual on April 7, 2022. Final analysis and correlative analyses are ongoing. Acknowledgements: Coordinated by the CCTG. Funding supported by SU2C Canada - Canadian Cancer Society Breast Cancer Dream Team Research Funding (SU2C-AACR-DT-18-15) and OICR. CFI-402257 provided by Treadwell Therapeutics. Citation Format: Philippe Bedard, Mihaela Mates, John Hilton, Nathalie Levasseur, Arif Awan, Amirrtha Srikanthan, David W. Cescon, Karen Gelmon, Andrew Robinson, Nancy Drummond-Ivars, Irene Li, Laleh Rastgou, Jackie Edwards, Linda Hagerman, Siwei Zhang, Mark Bray, Lesley Seymour, Moira Rushton, Pierre-Olivier Gaudreau. CCTG IND.236: A Phase 1b trial of combined CFI-402257 and weekly paclitaxel in patients with HER2-negative (HER2-) advanced breast cancer (aBC) [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-07-10.

Abstract CCTG IND.239: A phase 2 study of combined CFI-400945 and durvalumab in patients with advanced triple negative breast cancer (aTNBC) John Hilton, David W. Cescon, Andrew Robinson, Sukhbinder Dhesy-Thind, Sara Kristina Taylor, Arif Awan, Terry Ng, Moira Rushton, Marie-France Savard, Lindsay Muyot, Marie Claude Reeves, Linda Hagerman, Hongbo Lui, Mark Bray, Dongsheng Tu, Lesley Seymour, Pierre-Olivier Gaudreau Background: CFI-400945 is a selective oral inhibitor of Polo-like Kinase 4 (PLK4), which controls centriole duplication and mitotic progression, and was identified as a drug target based on functional screening of genomically unstable breast cancers. CFI-400945 monotherapy has anti-proliferative activity and enhances antitumor activity when combined with anti-PD-1 immune checkpoint blockade in transplantable murine cancer models. Material and methods: In this multi-centre phase II trial of CFI-400945 and durvalumab combination therapy, the primary objective was overall response rate (ORR) per RECIST 1.1. Patients with aTNBC with adequate organ function, PS 0-1, previously treated with >1 line of chemotherapy including anthracycline and/or taxane, were eligible. CFI-400945 32mg monotherapy was administered on a 7-day on, 7-day off schedule for cycle 1 (which reduced the likelihood of significant hematologic toxicity). From cycle 2 onwards, CFI-400945 32mg daily was administered in combination with durvalumab 1500mg IV every 28 days; responses were assessed every 8 weeks. Following trial activation, 3 patients received a CFI-400945 dose of 40mg (same schedule) for a total of 9 cycles before 32mg was declared as the new recommended phase 2 dose (based on other ongoing trials using CFI-400945). A Simon 2-stage design was used; ≥3/15 responses in stage 1 were required to expand to stage 2. Exploratory PD-L1 expression was measured on immune and tumor cells using the SP263 assay. Results: 15 patients received a total of 45 cycles (1-12 cycles per patient). Median age was 56 (31-76); 53% PS1; 20% 3 prior chemotherapy lines and; 27% 4 sites of metastatic disease. Immune vs tumor cell PD-L1 expression was 1% in 50% and 23% of patients, respectively (immune and tumor cell expression was mutually exclusive). Immune vs tumor cell PD-L1 expression was 10% in 17% and 15% of patients, respectively. Grade 3 hematological adverse events (AEs) were lymphopenia (40%), neutropenia (20%), anemia and thrombocytopenia (7% for both). One serious AE at least possibly related to treatment was seen: grade 3 febrile neutropenia. Frequent AEs (˃5%) considered at least possibly related to CFI-400945 were: nausea and anorexia (both 20%), fatigue and dysgeusia (both 13%), headache, dizziness, maculo-papular rash, back pain and gastroesophageal reflux disease (all 7%), Frequent AEs (˃5%) considered at least possibly related to durvalumab were: anorexia (13%), arthritis, fatigue, back pain, pain in extremity and hot flashes (all 7%). No responses were observed in 14 evaluable patients during stage 1, therefore the pre-specified threshold for anti-tumor activity to proceed to stage 2 was not met. Disease control rate (complete response, partial response or stable disease ˃16 weeks in duration) was 7% (1/14). Conclusions: CFI-400945 and durvalumab was well tolerated, with no unexpected toxicities of the combination. However, in this heavily pretreated and PD-L1 unselected TNBC population, no responses were observed and the pre-specified threshold for anti-tumor activity for stage 2 was not met. The trial was closed to accrual on April 26, 2022. Final analysis and correlative analyses are ongoing. Acknowledgements: Coordinated by the CCTG. Funding supported by Astra Zeneca. CFI-400945 provided by Treadwell Therapeutics and durvalumab provided by Astra Zeneca. Citation Format: John Hilton, David W. Cescon, Andrew Robinson, Sukhbinder Dhesy-Thind, Sara Taylor, Arif Awan, Terry L. Ng, Moira Rushton, Marie-France Savard, Lindsay Muyot, Marie Claude Reeves, Linda Hagerman, Hongbo Lui, Mark Bray, Dongsheng Tu, Lesley Seymour, Pierre-Olivier Gaudreau. CCTG IND.239: A phase 2 study of combined CFI-400945 and durvalumab in patients with advanced triple negative breast cancer (aTNBC) [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-07-18.

Background: TTK (also known as Mps1), a dual-specificity serine-threonine kinase, is critical for the spindle assembly checkpoint (SAC), chromosome alignment and error correction in mitosis. Inhibition of TTK causes premature mitotic exit with unattached chromosomes, resulting in chromosomal missegregation, aneuploidy and cell death. CFI-402257 is a potent (Ki = 0.09 nM, IC50 = 1.2 nM), highly selective and orally active inhibitor of TTK. Robust suppression of tumor growth was achieved upon oral dosing of single agent CFI-402257 in ER+/HER2- and triple negative breast cancer (TNBC) cell line and patient derived xenograft models. CFI-402257 demonstrated enhanced cytotoxicity in CDK4/6 inhibitor resistant ER+ breast cancer cell line models compared to parental cell lines, including those with RB1 loss. Methods: This multi-center Phase I dose escalation study (3+3 design) was designed to determine the safety, tolerability and maximum tolerated dose (MTD) of CFI-402257 and evaluate anti-tumor activity at the RP2D. CFI-402257 was dosed once daily on a continuous schedule in 28-day cycles at a starting dose of 5mg based on preclinical toxicology. Dose escalation included patients with advanced solid tumors and dose expansion at the RP2D into three expansion cohorts - Cohort A advanced solid tumors, Cohort B advanced ER+ or TNBC with 1-4 prior lines of chemotherapy for metastatic disease and Cohort C ER+/HER2- breast cancer in combination with Fulvestrant (500mg IM Day 1, 15 and 29 and then every 28 days) who have had prior treatment with an aromatase inhibitor in combination with a CDK4/6 inhibitor (>= 3 months) and =<1 prior chemotherapy for metastatic disease. Results: As of May 25, 2021, 66 patients had been enrolled, majority (76%) of patients received >3 prior therapies before study entry and 37% of patients’ (23/66) primary malignancy was breast cancer. The maximum administered dose was 294 mg and the study has continued enrolling at the recommended phase 2 dose of 168 mg with grade 3 Neutropenia, grade 3 Febrile Neutropenia and grade 3 Colitis as the dose limiting toxicities. Only 1/6 patients experienced a DLT at 168 mg (grade 3 Neutropenia, >7 days) The most common treatment emergent AEs (Gd3, >5%) were Neutropenia (15.6%), Anaemia (7.8%), Hypophosphataemia and Febrile Neutropenia (6.3%). PR’s confirmed by the Investigator have been seen to date in 33% of the cohort C subjects (2/6 patients), with an additional two cPR’s seen in the breast cancer population (ER+/HER2-) from the expansion cohorts. Conclusion: CFI-402257 is generally well tolerated and continues to enroll at 168mg daily with a manageable AE profile and early signs of anti-tumor activity. Enrollment in the expansion cohorts is ongoing and updated safety and efficacy data for the previously treated ER+/HER2- population will be presented at the time of the meeting. A multi-center phase II clinical trial is planned. Citation Format: John Hilton, Daniel J Renouf, David W Cescon, Aaron R Hansen, Albiruni RA Razak, Lee-Anne Stayner, Trisha Denny, Graham Fletcher, Tak W Mak, Mark Bray, Philippe L Bedard. Phase I study of cfi-402257, an oral ttk inhibitor, in patients with advanced solid tumors with breast cancer expansion cohorts [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-18-17.

TPS2619 Background: TTK (MPS1), a dual-specificity serine-threonine kinase, is critical for the spindle assembly checkpoint (SAC), chromosome alignment and error correction in mitosis. Inhibition of TTK causes premature mitotic exit with unattached chromosomes, resulting in chromosomal missegregation, aneuploidy and cell death. TTK is overexpressed in several tumor types, which may contribute to survival and proliferation of aneuploid cells, and higher expression correlates with adverse outcomes. The Campbell Family Therapeutics Group at the University Health Network (UHN) has developed CFI-402257, a potent (Ki = 0.09 nM, IC50 = 1.2 nM), highly selective and orally active inhibitor of TTK, with negligible activity towards 265 other kinases. Robust suppression of tumor growth was achieved upon oral dosing of single agent CFI-402257 at tolerated doses in several cell line (breast, colorectal) and patient-derived (ovarian) xenograft models. Pharmacodynamic effects including reduction in phospho-histone H3 were observed. In syngeneic mouse colorectal cancer models, CFI-402257 + PD-1 immune checkpoint blockade demonstrated greater activity than either agent alone, and resulted in tumor regressions and immunity to rechallenge. Methods: This multi-center Phase I dose escalation study (3+3 design) will determine the safety, tolerability and maximum tolerated dose (MTD) of CFI-402257 administered as daily continuous oral treatment. Secondary and correlative endpoints include plasma PK, antitumor activity, and molecular features associated with response or clinical benefit. An expansion cohort (n = 12) will be enrolled at the MTD. Key inclusion criteria: adult patients with advanced solid tumors, measurable disease (RECIST 1.1), adequate organ function and performance status (ECOG 0-1). Exclusion criteria: uncontrolled medical illness, CNS metastases (unless stable x 3 months). CFI-402257 will be dosed once daily on a continuous schedule in 28-day cycles, beginning at 5 mg/day with planned escalation to 56 mg/day. DL1 completed enrolment 01/2017 and accrual is ongoing. Phase II studies are planned (Stand Up to Cancer Canada Breast Cancer Dream Team). Funding: UHN, CIRM. Clinical trial information: NCT02792465.

Abstract Abstract 3307 Background: ENMD-2076 is a novel, orally-active molecule that inhibits Aurora A kinase and FLT3, as well as multiple receptor tyrosine kinases that drive tumor vascularization, including VEGFR2 (KDR), PDGFR and FGFR. A phase I study was conducted to determine the maximum tolerated dose (MTD) and toxicities of ENMD-2076 in patients with refractory hematological malignancies. Methods. Patients (cohorts of 6 evaluable patients per dose level) received escalating doses of ENMD-2076 administered orally daily [225 mg (n=7), 375 mg (n=6), 325 mg (n=9), or 275 mg (n=5)]. Plasma levels of ENMD-2076 were measured on Days 1, 8, & 29 of cycle 1 and at the end of study. Peripheral blood and/or bone marrow were obtained at baseline for ex vivo drug sensitivity testing, and on Days 8 & 29 of cycle 1 for pharmacodynamic (PD) monitoring to assess the effects of ENMD-2076 on cell signaling pathways. The latter flow cytometry-based assay used combined pERK, pAkt, pSTAT5, and pS6 labelling, and tested the effects of acute stimulation with SCF and FLT3 ligands in the presence or absence of pathway inhibitors, including ENMD-2076. Results. Twenty-seven patients have been treated to date (26 AML; 1 CMML-2). Median age is 69 years (range, 43–84 years). Median ECOG status 1 (range, 0 to 2). Eighteen patients (89%) had received prior therapy (median, 2 regimens; range, 0 to 6 regimens); 2 had received a prior allogeneic stem cell transplant. A total of 42 cycles have been administered, with a median of 1 cycle (range, 0 to 8 cycles); 10 patients (37%) have received 2 or more cycles of therapy. The most common nonhematological toxicities of any grade, regardless of association with drug, were fatigue, diarrhea, dysphonia, dyspnea, hypertension, constipation, and abdominal pain. Two of 6 patients treated at the 375 mg/day dose level developed dose-limiting toxicities (DLT), consisting of grade 3 fatigue. The dose was therefore decreased to 325 mg/day; one patient on this dose level developed a DLT with grade 3 fatigue. However, as 2 additional patients subsequently developed grade 2 or 3 fatigue (during cycles 1 and 2, respectively), the 325 mg/day dose was not believed to be tolerable for chronic administration in this patient population. The dose was therefore decreased to 275 mg/day; 2 patients subsequently developed DLTs consisting of grade 3 typhilitis and grade 3 syncope, respectively. Overall, no patient experienced grade 4 toxicities or death from ENMD-2076. Of the 20 evaluable patients, one patient achieved a CRi (transfusion-independent with platelets <100 × 109/L), three a morphologic leukemia-free state (MLFS) with a major HI-P and/or HI-E, and 4 other patients had an 11%, 14%, 23%, and 65% reduction in marrow blast count, respectively. Ex vivo studies showed that pre-incubation with ENMD-2076 in concentrations in the range of 0.5–2 mM suppressed growth factor stimulation in the blast cells of most patients tested to date. Decreases in the stimulation of ERK, Akt, STAT5, and S6 were seen in the Day 8 & 29 samples, including a striking inhibition of cell signaling in one patient who achieved a CRi. Conclusions. Single agent ENMD-2076 has activity in a heavily pretreated group of AML patients that may correlate with inhibition of ERK, Akt, STAT5, and S6 activity. The recommended phase two dose (RPTD) is 225 mg/day. Enrollment, as well as PK and PD monitoring of this study, is ongoing. Disclosures: Off Label Use: Clinical trial investigating ENMD-2076 in patients with relapsed or refractory leukemia. Sidor:EntreMed, Inc.: Employment. Fletcher:EntreMed, Inc.: Employment. Arnott:EntreMed, Inc.: Employment. Bray:EntreMed, Inc.: Employment.

AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO MKC-1 is a clinical-stage orally active cell-cycle inhibitor that exhibits antiproliferative activity through G2/M arrest, apoptosis, and antiangiogenic effects. MKC-1 binds to tubulin and inhibits microtubule formation and, in addition, binds to members of the importin-\#946; family. Binding has been shown by the property of induced fluorescence, whereby MKC-1 exhibits no appreciable fluorescence properties in aqueous solution but fluoresces intensely when dissolved in organic solvents or when bound by target proteins. Here, MKC-1 induced fluorescence has been exploited to determine the binding site of the compound to target proteins. MKC-1 was shown to bind to the colchicine site of tubulin, through competition with colchicine and other colchicine-site binding drugs such as 2-methoxyestradiol and combretastatin A4, and the absence of competition with vincristine. Importin-\#946;s belong to a superfamily of proteins containing HEAT-repeats, which are tandemly repeated sequences with helical topology approximately 50 amino acids in length. The MKC-1 binding region of importin-\#946;1 was determined by taking advantage of this modular architecture and generating GST-tagged recombinant fragments of the protein. MKC-1 binds to the C-terminal portion of importin-\#946;1, between residues 500-876, as a GST-tagged fragment comprised of these residues induced MKC-1 fluorescence at 1.3-fold the intensity of the full-length protein, whereas recombinant fragments not containing these residues induced MKC-1 fluorescence at levels 0.25-fold or lower of that of full-length GST-importin-\#946;1. Furthermore, these results, along with fluorescence titration experiments, show that MKC-1 binds to importin-\#946;1 at a single, distinct site, despite importin-\#946;1 being comprised of 19 HEAT-repeat units. Primary sequence homology amongst HEAT-repeat family members suggested that MKC-1 may bind other HEAT-repeat proteins, including the key oncological target mammalian target of rapamycin (mTOR). Indeed, MKC-1 does inhibit signaling through the PI3K-Akt-mTOR signaling pathway. Analogous to the importin-\#946;1 experiments, the binding of MKC-1 to mTOR is being investigated through recombinant fragments of the protein; MKC-1 unequivocally does not bind to the FK506/rapamycin binding domain of mTOR. The fluorescence properties of MKC-1 are also exploited to determine the cellular localization of the compound, suggesting the nature of intracellular targets. This property of induced fluorescence could also be used to search for new MKC-1 targets. Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 5353.

Abstract ENMD-2076 is a novel, orally-active molecule that inhibits Aurora A kinase as well as multiple receptor tyrosine kinases that drive tumor vascularization, including VEGFR2 (KDR), PDGFR and FGFR. A phase 1 study was conducted to determine the maximum tolerated dose (MTD) and toxicities of ENMD-2076 in patients with refractory hematological malignancies. Fifteen patients with AML (cohorts of 6 patients per dose level) have been treated with 225 (n=7), 325 (n=2), and 375 (n=6) mg of ENMD-2076 administered orally once daily. Median age was 76 years (range, 60 to 82 years). Median ECOG status was 1 (range, 0 to 2). Fourteen patients had received prior therapy (median, 2 regimens; range, 0 to 4 regimens). A total of 17 cycles have been administered to date, with a median of 1 cycle (range, 0 to 3 cycles); 2 patients (14%) have received 3 or more cycles of therapy. The most common ENMD-2076-related adverse events were grade ≤ 2 and consisted of dizziness, petechiae, hypertension, nausea, fatigue, diarrhea, and reflux. Dose-limiting toxicity consisted of grade 3 fatigue in 2 patients at the 375 mg/day dose level. Therefore, the dose was decreased to 325 mg/day. Following a drug holiday, both patients who had experienced the grade 3 fatigue were restarted at the 325 mg/day dose level. No patient experienced grade 4 toxicities or death from ENMD-2076. Of the 13 evaluable patients, 1 patient achieved a morphologic leukemic free state (MLFS) with platelet transfusion independence. One patient achieved a HI-P. Two other patients had a 12% and 14% reduction in marrow blast count, respectively. At the time of analysis, 3 patients discontinued therapy due to disease progression. Peripheral blood and/or bone marrow were obtained at baseline for ex vivo drug sensitivity testing, and on Days 8 and 29 of cycle 1 for pharmacodynamic (PD) monitoring, using a whole blood flow cytometry protocol to measure ENMD-2076 effects on cell signalling pathways. This assay uses combined labelling for P-ERK, P-Akt, P-STAT5, and P-S6 as the readout, and tests the effects of acute stimulation with the ligands SCF and FL in the presence or absence of pathway inhibitors, including ENMD-2076. Pre-incubation with drug concentrations in the range 0.5 – 2 µM suppressed growth factor stimulation in the blast cells of all patients tested to date. Decreases in the ability to stimulate ERK,Akt, STAT5, and S6 were seen in the Day 8 and 29 samples, including a striking inhibition of cell signalling in one patient who achieved a MLFS. Assays are also in progress to monitor specific effects on Aurora kinase and the cell cycle in these patient samples. In conclusion, single agent ENMD-2076 has activity in a heavily pretreated group of AML patients that may correlate with inhibition of ERK,Akt, STAT5, and S6 activity. Enrollment, as well as PK and PD monitoring of this study, is ongoing. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A106.