Hepatocellular carcinoma (HCC) remains a major cause of cancer mortality worldwide, largely due to its high recurrence rate and chemoresistance. The microtubule-associated protein regulator of cytokinesis 1 (PRC1) has been implicated in HCC progression, associated with poor prognosis, enhanced cancer proliferation, stemness, metastasis, and chemoresistance. PRC1 functions as an oncogenic driver, particularly in HCC cases with elevated expression, where it promotes Wnt signaling and the expression of genes linked to early recurrence. BKT300, a novel targeted anti-cancer agent, was found to inhibit PRC1, offering a potential treatment option for advanced, difficult-to-treat cancers that express high levels of PRC1, such as HCC. This study investigated the effects of BKT300 on HCC cell migration and survival pathways. BKT300 selectively inhibited HCC cell migration, induced G2/M cell cycle arrest, and triggered mitotic catastrophe and apoptosis through the caspase 3 pathway. BKT300 targeted PRC1 by arresting it in a phosphorylated state at T481, concurrently downregulating CDC25C and upregulating p21. siRNA-mediated suppression of PRC1 in HCC cells prevented the activity of BKT300, indicating that BKT300’s efficacy is dependent on PRC1 expression. Direct binding of BKT300 to PRC1 was further confirmed through multiple assays. In vivo studies in two HCC mouse models demonstrated significant tumor growth inhibition and tumor regression, underscoring the therapeutic promise of BKT300 for HCC. Overall, BKT300 presents a novel approach to tackling HCC by directly targeting PRC1, potentially improving outcomes in HCC cases with high PRC1 expression and resistance to conventional therapies. Amnon Peled, Lika Gamaev, Hanna Wald, Rakefet Rosenfeld, Orly Eizenberg, Arnon Aharon, Michal Abraham. Bkt300, a novel small molecule targeting the protein regulator of cytokinesis 1 pathway in hepatocellular carcinoma [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 6874.

Protein regulator of cytokinesis 1 (PRC1) is frequently overexpressed in various cancers and is associated with poor prognosis. BKT300 is a small molecule shown to selectively inhibit leukemic cell migration and survival by targeting the PRC1 pathways. The current work aimed to examine the role of PRC1 in acute myeloid leukemia (AML) and to assess the impact of BKT300, a small molecule PRC1 inhibitor, on AML cell viability and tumor growth in mouse xenograft AML models. BKT300 directly bound PRC1, resulting in disrupted actin and microtubule formation, G2/M cell cycle arrest, mitotic catastrophe and apoptosis via the caspase-3 pathway in AML cells. BKT300 inhibited PRC1 dephosphorylation at T481, downregulated CDC25C and upregulated p21, effectively halting the cell cycle and inhibiting leukemic cell proliferation while sparing normal cells. PRC1 was found to be overexpressed in AML patients and cell lines, with high levels associated with reduced overall patient survival. In addition, PRC1 expression levels correlated with BKT300 efficacy. BKT300 treatment led to 98% of tumor growth inhibition and 89.4% of tumor regression in mouse xenograft AML models, without notable impacts on normal hematopoiesis or biochemistry, even at high doses. As a first-in-class targeted therapy, BKT300 presents a promising new treatment option for advanced AML.

Pancreatic cancer (PDAC) is a major cause of cancer mortality worldwide, largely due to its high recurrence rate and chemoresistance. We found that the microtubule-associated protein regulator of cytokinesis 1 (PRC1) is implicated in PDAC progression, associated with poor prognosis, enhanced cancer proliferation, stemness, metastasis, and chemoresistance. PRC1 functions as an oncogenic driver, particularly in PDAC cases with elevated expression. Using siRNA and the small molecule BKT300 that inhibits PRC1 function, we found that downmodulation of PRC1 induces G2/M cell cycle arrest and triggered mitotic catastrophe and apoptosis. Interestingly, BKT300 targeted PRC1 by arresting it in a phosphorylated state at T481, concurrently downregulating CDC25C and upregulating p21. siRNA-mediated suppression of PRC1 in cancer cells prevented the activity of BKT300, indicating that BKT300’s efficacy is dependent on PRC1 expression. Direct binding of BKT300 to PRC1 was further confirmed through multiple assays. Importantly, BKT300 synergized with 5FU and irinotecan to induce cancer cell death, both agents are the standard of care for treatment of pancreatic cancer patients. In vivo studies in PDAC mouse models demonstrated significant tumor growth inhibition and tumor regression, underscoring the therapeutic promise of BKT300 for PDAC. Overall, BKT300 presents a novel approach to tackling PDAC by directly targeting PRC1, potentially improving outcomes in PDAC cases with high PRC1 expression and resistance to conventional therapies. Amnon Peled, Michal Abraham, Lika Gamaev, Orly Eizenberg, Arnon Aharon. Targeting the Protein Regulator of Cytokinesis 1 (PRC1) Pathway in Pancreatic Cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research—Emerging Science Driving Transformative Solutions; Boston, MA; 2025 Sep 28-Oct 1; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_3):Abstract nr A056.

Abstract Aberrant migration and proliferation of cancer cells are the hallmark of metastatic tumors. Protein Regulator of Cytokinesis 1 (PRC1) is a scaffold protein that regulates metabolic pathways of cancer cell proliferation, survival, and metastasis. BKT300 is a small molecule (MW 399.33, C22H25NO6), that selectively inhibits the migration and survival of hematological and solid cancer cells. BKT300 inhibits, at high affinity, both actin and microtubule formation and organization leading to G2/M cell cycle arrest, mitotic catastrophe, and apoptosis through the caspase 3 pathway. These effects are selective to cancer cells and were not observed on normal cells. BKT300 was found to arrest PRC1 in a phosphorylated form at position T481, down regulates the expression of CDC25C and up regulate p21. Suppression of PRC1 using SiRNA ablation inhibited BKT300 induced apoptosis on several tumor cell lines and inhibited the effect of BKT300 on G2M cell cycle arrest. Furthermore, using Biacore SPR, microscale thermophoresis (MST), and ELISA assays we were able to demonstrate the direct binding of BKT300 to PRC1. Beyond its single agent activity, BKT300 was shown to synergize with standard of care (SoC) treatments, including anti PD-1 immunotherapy, BCL-2 inhibitor (Ventoclax), topoisomerase I inhibitor (Irinotecan) topoisomerase II inhibitor (Doxorubicin), anti-metabolite Fluorouracil (5-FU) and alkylating agent (Temozolomide). BKT300 has a remarkable safety profile when administered at high doses to mice rats, minipigs and non-human primates. Treatment with BKT300 showed almost no effects on hematopoiesis and biochemistry in doses far higher than the affective ones. The in-vivo activity of BKT300 was demonstrated in multiple Xenograft, Syngeneic and PDX models in mice. In-vivo treatment with BKT300 resulted in preventive (small tumors) as well as therapeutic (large tumors) activity in triple negative breast cancer (TNBC) xenograft, MDA-MB-453 and MDA-MB-468 models leading to over then 70%-90% reduction in tumor size following one to two treatments cycles. Similar results were obtained using PDX models derived from patients with Colon, Ovarian and Pancreatic cancer. BKT300 is a novel, first in class, targeted anti-cancer treatment that inhibits the activity of the scaffold protein PRC1. BKT300 is expected to provide a new treatment option to, the high unmet medical need of, advanced difficult to treat cancers, specifically ones that overexpress PRC1 and CDC25C. Citation Format: Amnon Peled, Michal Abraham, Hanna Wald, Rakefet Rosenfeld, Orly Eizenberg, Arnon Aharon. Novel anti-cancer small molecule targeting the protein regulator of cytokinesis pathway [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 674.

<p>Expression levels of genes in CXCR4 overexpressing cell lines</p>

PDF file - 132K, Supplementary Table 1. Demographic characteristics of human patients. Supplementary Table 2. History of MM Therapy/Chemotherapy. Supplementary Table 3. Time from BKT140 dosing to first Stem cell (CD34+) collection and the number of stem cells collected at the first aphaeresis following administration of BKT140. Supplementary Table 4. Number of WBC and CD34+ cells in the blood pre dosing and post dosing with BKT140. Supplementary Table 5. Inclusion Criteria.

Supplementary Figure 2 - PDF file 64K, CXCR4 migratory function in NHL cells

<p>Expression levels of genes in CXCR4 overexpressing cell lines</p>

<p>CXCL12 levels of NB cells</p>

<p>BCL2 levels in SKNBE-CXCR4 cells</p>

Supplementary Figure 3 - PDF file 100K, Apoptosis induced by BKT140 and Rituximab

Supplementary Figure 4 - PDF file 117K, Interaction of NHL cells with BMSCs

miR-15a and miR-16-1 expression following antagomiR transfection

Supplementary Figure 5 - PDF 65K,Apoptosis induction in BL-2 cells infiltrated to the BM

PDF - 90K, Demonstrates CXCR4 expression and function in K562 and K562LG-CXCR4 cells.

Supplementary Table S1A: Part 1 Summary of Baseline Demographic Characteristics Supplementary Table S1B: Part 2 Summary of Baseline Demographic Characteristics

Supplemented Table 4A: Absolut number of different subsets of hematopoietic stem cells Supplemented Table 4B: Percentages of different subpopulations of BL-8040 mobilized cells as compared to G-CSF mobilized cells

Drug-related adverse events by preferred term and treatment group

Table S2A: Summary Statistics of Pharmacokinetic Parameters of BL-8040 after Day 1 (First Dose) in Healthy Volunteers. Table S2B: Summary Statistics of Pharmacokinetic Parameters of BL-8040 after Day 2 (Second Dose) in Healthy Volunteers