Di Tian

Supplementary Figure 2. Randomization schemes to assign treatment cohorts, based on mouse body weight and whole head total photon flux.

Breast cancer brain metastasis (BCBM) remains a clinical challenge marked by limited therapeutic options and poor survival rates. Approximately 30% of all patients with metastatic breast cancer develop BCBM, with the highest incidence in patients with aggressive molecular subtypes, including triple-negative breast cancer (TNBC). Patients with TNBC with brain metastasis experience rapid disease progression and significantly reduced survival times due to a lack of targeted treatments that can penetrate the blood–brain barrier (BBB) and effectively control metastatic expansion. Current treatment options, such as whole-brain radiotherapy and chemotherapy, offer limited efficacy and are associated with significant toxicities, underscoring the urgent need for novel therapeutics that can target BCBM directly. We developed innovative colchicine binding site inhibitors (CBSI) targeting tubulin, SB-216 and SP-1-39, that show potent preclinical efficacy against brain and extracranial metastases in BCBM models. Both CBSIs cross the BBB, inhibit cell growth and migration, and induce apoptosis with low nanomolar potencies, similar to Azixa (MPC-6827), another CBSI previously evaluated in clinical trials. SB-216 reduced brain and concurrent extracranial metastases in a preventive dosing paradigm, extending overall survival. SB-216 also suppressed the expansion of preestablished brain lesions. In a taxane-refractory patient-derived TNBC model, SP-1-39 therapy markedly reduced brain and extracranial tumor burden. Together, these results highlight the promising therapeutic potential of SB-216 and SP-1-39 in treating taxane-sensitive or -resistant BCBM, filling a critical gap in TNBC management by offering targeted treatments that can cross the BBB and combat chemorefractory disease.

Supplementary Figure 1. SB-216 and SP-1-39 potently inhibit tumor cell growth in TNBC TxR sublines, and TxR cells up-regulate P-gp (ABCB1) expression at the mRNA and protein levels.

Supplementary Figure 4. Maximum tolerated dose (MTD) study for SP-1-39 HCl salt when administered by intraperitoneal (IP) injection.

Supplementary Figure 6: Brain vacuolization was observed in all mice with end-stage brain metastases.

Supplementary Figure 3. Bioimaging data for example mice from the SB-216 overall survival study through day 21 of treatment.

Supplementary Figure 5: No gross changes in pathology were observed between vehicletreated and CBSI-treated mice at treatment study endpoints.

Supplementary Table 1. The BBB score, which accounts for multiple structural properties of compounds to predict the likelihood of activity in the central nervous system, was calculated in the Schrodinger Modeling software using the equation described in (50): ∑T0 [Aro_R, HA, (1.5) MWHBN, 2 (TPSA), (0.5) pka].

Breast cancer brain metastasis (BCBM) remains a clinical challenge marked by limited therapeutic options and poor survival rates. Approximately 30% of all patients with metastatic breast cancer develop BCBM, with the highest incidence in patients with aggressive molecular subtypes, including triple-negative breast cancer (TNBC). Patients with TNBC with brain metastasis experience rapid disease progression and significantly reduced survival times due to a lack of targeted treatments that can penetrate the blood–brain barrier (BBB) and effectively control metastatic expansion. Current treatment options, such as whole-brain radiotherapy and chemotherapy, offer limited efficacy and are associated with significant toxicities, underscoring the urgent need for novel therapeutics that can target BCBM directly. We developed innovative colchicine binding site inhibitors (CBSI) targeting tubulin, SB-216 and SP-1-39, that show potent preclinical efficacy against brain and extracranial metastases in BCBM models. Both CBSIs cross the BBB, inhibit cell growth and migration, and induce apoptosis with low nanomolar potencies, similar to Azixa (MPC-6827), another CBSI previously evaluated in clinical trials. SB-216 reduced brain and concurrent extracranial metastases in a preventive dosing paradigm, extending overall survival. SB-216 also suppressed the expansion of preestablished brain lesions. In a taxane-refractory patient-derived TNBC model, SP-1-39 therapy markedly reduced brain and extracranial tumor burden. Together, these results highlight the promising therapeutic potential of SB-216 and SP-1-39 in treating taxane-sensitive or -resistant BCBM, filling a critical gap in TNBC management by offering targeted treatments that can cross the BBB and combat chemorefractory disease.