Dose-dependent Tumor Growth Inhibition Research Articles

Identification of a novel potent CDK inhibitor degrading cyclinK with a superb activity to reverse trastuzumab-resistance in HER2-positive breast cancer in vivo

CDK12 is overexpressed in HER2-positive breast cancers and promotes tumorigenesis and trastuzumab resistance. Thus CDK12 is a good therapeutic target for the HER2-positive breast tumors resistant to trastuzumab. We previously reported a novel purine-based CDK inhibitor with an ability to degrade cyclinK. Herein, we further explored and synthesized new derivatives, and identified a new potent pan-CDK inhibitor degrading cyclinK (32e). Compound 32e potently inhibited CDK12/cyclinK with IC50 = 3 nM, and suppressed the growth of the both trastuzumab-sensitive and trastuzumab-resistant HER2-positive breast cancer cell lines (GI50's = 9–21 nM), which is superior to a potent, clinical pan-CDK inhibitor dinaciclib. Moreover, 32e (10, 20 mg/kg, ip, twice a week) showed a dose-dependent inhibition of tumor growth and a more dramatic anti-cancer effect than dinaciclib in mouse in vivo orthotopic breast cancer model of trastuzumab-resistant HCC1954 cells. Kinome-wide inhibition profiling revealed that 32e at 1 μM exhibits a decent selectivity toward CDK-family kinases including CDK12 over other wildtype protein kinases. Quantitative global proteomic analysis of 32e-treated HCC1954 cells demonstrated that 32e also showed a decent selectivity in degrading cyclinK over other cyclins. Compound 32e could be developed as a drug for intractable trastuzumab-resistant HER2-positive breast cancers. Our current study would provide a useful insight in designing potent cyclinK degraders.

Abstract A090: Preclinical efficacy of BDTX-4933, a brain-penetrant, orthosteric RAF inhibitor, targeting oncogenic RAF conformation shared by groups of BRAF and upstream driver mutations

Abstract Alterations in the RAS-MAPK pathway, such as mutations in NF1, RAS, and BRAF, often lead to oncogenic signaling and result in aberrant cell proliferation and tumor growth. Oncogenic BRAF mutations may be activated as either monomers or dimers, while oncogenic RAS mutations or loss-of-function mutations in NF1, a negative regulator of RAS, drive activation of RAF dimers to transduce MAPK signaling. Currently approved BRAF inhibitors are selective against monomeric BRAF mutations and largely inactive against dimeric RAF mutants. Additionally, these agents may also promote deleterious paradoxical activation of RAF in select contexts. Moreover, FDA-approved KRAS G12C mutant-selective inhibitors are ineffective for cancer patients who harbor other (non-G12C) KRAS, NRAS, and NF1 mutations which all lead to active RAF dimer. There remains a high unmet clinical need for a highly CNS penetrant RAF inhibitor that targets a broad spectrum of oncogenic RAF conformations in the context of RAF, RAS, and other upstream driver mutations. BDTX-4933 is a next generation MasterKey RAF inhibitor designed to address the shortcomings of previous generation RAF inhibitors. BDTX-4933 is an orthosteric inhibitor that potently targets the oncogenic RAF conformation shared by groups of RAF driver mutations and induces an inactive conformation of RAF. As a MasterKey inhibitor, BDTX-4933 potently inhibits a broad spectrum of BRAF mutations and active RAF dimers promoted by upstream oncogenic MAPK pathway alterations, such as KRAS, NRAS, and NF1 mutations. In vivo, once daily oral dosing of BDTX-4933 resulted in robust and sustained target engagement, inhibiting ERK phosphorylation without inducing paradoxical activation. In a mouse breadth of efficacy study with patient derived xenografts (PDX), BDTX-4933 demonstrated tumor growth inhibition and regression as a single agent across models expressing a variety of targeted BRAF or MAPK pathway oncogenic mutations. Additionally, BDTX-4933 as a single agent and in combination with a MEK inhibitor, demonstrates anti-tumor efficacy in KRAS mutant lung PDX models. In a PDX model of BRAF V600E positive tumor that progressed following BRAF and MEK combination therapy, BDTX-4933 achieved robust tumor growth regression. Furthermore, BDTX-4933 exhibits high CNS exposure leading to dose-dependent tumor growth inhibition, and survival benefit in mice implanted intracranially with xenograft BRAF mutant tumors. BDTX-4933 has a best-in-class RAF inhibitor profile to treat cancer patients harboring BRAF mutations or RAF dimer-promoting upstream genetic alterations. BDTX-4933 is currently in a Phase I clinical study in patients with solid tumor harboring sensitive BRAF and RAS alterations (NCT05786924). For more information on this active Phase 1, please contact: ClinicalTrials@bdtx.com. Citation Format: Yoon-Chi Han, Pui Yee Ng, Luisa Shin Ogawa, Shao Ning Yang, Miao Chen, Darlene Romashko, Tai-An Lin, Elizabeth Buck. Preclinical efficacy of BDTX-4933, a brain-penetrant, orthosteric RAF inhibitor, targeting oncogenic RAF conformation shared by groups of BRAF and upstream driver mutations [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A090.

Abstract PR011: Discovery of VVD-065, a first-in-class allosteric molecular glue of the Keap1-Cul3 E3-ligase complex for the treatment of NRF2-activated cancers

Abstract Somatic gain of function mutations in nuclear factor erythroid 2-related factor 2 (NRF2) transcription factor or loss of function mutations in Kelch-like ECH Associated Protein 1 (KEAP1) E3 ligase, which regulates NRF2 protein levels, are frequently identified in many solid cancers such as non-small cell lung cancer (NSCLC), esophageal squamous cell carcinoma (ESCC), and head and neck squamous cell carcinoma (HNSCC). These genomic alterations drive the activation of cytoprotective NRF2 transcriptional programs. In addition to mutational activation, NRF2 pathway activation has been documented with high frequency in patients without mutations in the pathway. Since tumor cells with hyperactivated NRF2 have been demonstrated to be dependent on this pathway for survival, targeting NRF2 represents an attractive therapeutic approach in tumors with aberrant NRF2 activation. Here, we report the identification of VVD-065, a first-in-class NRF2 inhibitor that acts via an unprecedented mechanism of action. Covalent modification of sensor cysteines on KEAP1 during electrophilic or oxidative stress is known to inhibit KEAP1 mediated NRF2 degradation. In complete contrast, covalent modification of a KEAP1 sensor cysteine by VVD-065 dramatically enhances NRF2 degradation. At low nM concentrations, VVD-065 covalently targets KEAP1 E3 ligase, and allosterically increases the affinity between KEAP1 and CUL3, thereby promoting the formation of active KEAP1-CUL3 E3 ligase complexes and increasing the rate of NRF2 degradation. VVD-065 profoundly reduces NRF2 protein levels in WT KEAP1/NRF2 settings, as well as various KEAP1 and NRF2 mutant settings. Consequently, VVD-065 substantially inhibits NRF2-dependent gene expression and proliferation of NRF2-dependent cell lines. In vivo, oral administration of VVD-065 results in robust degradation of NRF2 and decreased expression of NRF2 target genes. Tumor growth inhibition studies with cell-line derived, and patient derived NSCLC/ESCC xenograft (CDX/PDX) models revealed robust dose-dependent tumor growth inhibition and tumor regression in the absence of any overt toxicity. Since constitutive NRF2 activation often contributes to chemotherapeutic resistance, we also conducted TGI studies with 100+ PDX models to assess combination opportunities with chemotherapeutic agents. A marked combination response was achieved with cisplatin and nab-paclitaxel in various solid tumor types such as NSCLC, ESCC, HNSCC, and uterine cancers. Combination with chemotherapy was safe and well-tolerated in these mouse studies. In summary, we have identified VVD-065, a potent and selective KEAP1 activator that promotes the degradation of NRF2. Degradation of NRF2 led to robust monotherapy response in NRF2-activated cancers and also chemo-sensitized chemo-refractory tumors. A related molecule, acting through the same mechanism of action has now entered into Phase I clinical trials. Citation Format: Nil Roy, Tine Wyseure, I-Chung Lo, Jordon Inloes, Aaron Snead, Steffen Bernard, Justine Metzger, Jonathan Pollock, Stephanie Grabow, Christie Eissler, Melaminah Williams, Sarah Jacinto, Gabe Simon, Todd Kinsella, David Weinstein, Matt Patricelli. Discovery of VVD-065, a first-in-class allosteric molecular glue of the Keap1-Cul3 E3-ligase complex for the treatment of NRF2-activated cancers [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr PR011.

Abstract C145: ANS014004, a novel small-molecule type II c-Met inhibitor effectively overcomes clinical-resistance MET mutations and exhibits antitumor activity in preclinical models of MET-amplified non-small cell lung cancer (NSCLC) and gastric cancer

Abstract Mesenchymal epithelial transition (MET) proto-oncogene receptor tyrosine kinase (RTK) is a cell surface receptor selective for hepatocyte growth factor (HGF), and is involved in embryogenesis regulation, wound healing, organ regeneration, angiogenesis, and immunomodulation. Aberrant MET oncogenic alterations include: MET exon 14 skipping (MET∆ex14) mutations; activating mutations in the kinase domain; MET gene amplification; MET fusions; and MET protein overexpression. These oncogenic alterations occur in a wide range of human solid cancers. Small-molecule type I c-Met inhibitors bind to the active (Asp-Phe-Gly “DFG-in” state) of c-Met within the adenosine triphosphate (ATP) binding pocket via the Y1230 residue, resulting in down-regulation of multiple pro-oncogenic downstream factors. Presently, type I c-Met inhibitors such as Capmatinib are used as monotherapies in patients with locally advanced or metastatic NSCLC with MET∆ex14 mutations. However, development of post-treatment resistance to type I c-Met inhibitors occurs clinically, including through acquired mutations in codons D1228 and Y1230. No drugs have been approved globally for MET alteration indications other than MET∆ex14 mutations. Next generation MET inhibitors are thus needed to treat patients harboring various MET oncogenic alterations beyond MET∆ex14, including post-treatment acquired mutations. ANS014004 is a newly developed small-molecule type II inhibitor of c-Met, binding to the inactive state of c-Met (“DFG-out”) in the ATP pocket. ANS014004 showed greater kinase inhibition activity against c-Met mutant kinases Y1230H and D1228A in vitro (IC50’s = 2.596 and 2.971 nM, respectively), when compared with Capmatinib, Cabozantinib, and Merestinib (IC50 values = >5000, ~10, and ~5 nM respectively). Further, ANS014004 showed a stronger anti-proliferative effect in MET mutant Ba/F3 cells TPR-Met D1228A and TPR-Met-Y1230H in vitro (IC50’s = 49.34 nM and 30.32 nM, respectively), when compared with Capmatinib (IC50 values>10000nM) and Merestinib (IC50 values: 37~100 nM). ANS014004 also showed potent antitumor effects in vitro in the human cancer tumor cell lines harboring various pathogenetic MET alterations: Hs-746T (gastric); MKN-45 (gastric); EBC-1 (NSCLC); NCI-H1993 (NSCLC); with IC50 ranging from 6.38~62.32 nM. In several subcutaneous xenograft tumor model studies using these same cell lines implanted in BALB/c nude mice, orally administered ANS014004 produced significant dose-dependent tumor growth inhibition, and the corresponding exposures of ANS014004 in plasma, tumor and brain samples increased with increasing dose. ANS0104001 was also observed to induce tumor regression in a MET-amplified intracranial xenograft model. Overall, the non-clinical studies of ANS014004 demonstrated it is a potent, orally-bioavailable type II c-Met inhibitor with activity against various pathogenetic MET alterations and with favorable absorption, distribution, pharmacokinetic, efficacy, and tolerability profiles in vivo. The molecule will enter a Phase 1 clinical study in the US soon. Citation Format: Gong Li, Hepeng Shi, Peilong Zhang, Xiangqiu Li, Wenli Lan, Linna Li, David Chung, Kevin Schaab. ANS014004, a novel small-molecule type II c-Met inhibitor effectively overcomes clinical-resistance MET mutations and exhibits antitumor activity in preclinical models of MET-amplified non-small cell lung cancer (NSCLC) and gastric cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C145.

Abstract C085: OPN-6602, a dual EP300/CBP bromodomain inhibitor modulates androgen-driven transcription, including MYC, in mCRPC

Abstract Metastatic castration-resistant prostate cancer (mCRPC) remains an urgent unmet medical need despite existing therapies that target androgen receptor (AR) signaling. Disease progression is commonly a result of acquired resistance to existing anti-androgen therapies. Biological models have revealed potential resistance mechanisms of CRPC through the reactivation of the AR pathway. Transcriptional coactivators CREB-binding protein (CBP) and E1A binding protein (EP300) are high value targets in mCRPC as they mediate androgen-dependent and -independent activity of the AR pathway. EP300 and CBP proteins harbor both histone acetyltransferase (HAT)- and bromo-domains, and the HAT activity of EP300 regulates AR function. EP300 bromodomain inhibition partially blocks its HAT activity, reducing AR-mediated gene expression and tumor growth. OPN-6602 and OPN-6742 are potent, selective, and orally active small molecule dual EP300/CBP bromodomain inhibitors which abrogate persistent AR signaling in CRPC by modulating H3K27 acetylation and the recruitment of EP300, AR full length (AR-FL), and AR variant 7 (AR-v7) to AR response elements. OPN-6602 and OPN-6742 obstruct the EP300 bromodomain leading to downregulation of the AR pathway and consequent anti-tumor effects in AR-dependent CRPC. OPN-6602 and OPN-6742 are highly selective (>100-fold) against other bromodomains based on a screening panel of 40 bromodomains. Biochemical screens using AlphaScreen technology show these compounds to be equipotent against EP300 and CBP. OPN-6602 is particularly potent by Surface Plasmon Resonance analysis, displaying a KD of 0.87 nM, with a fast association rate (1.5 E+06/Ms) and a slow dissociation rate (1.3 E-03/s). Consequently, OPN-6602 displays a noteworthy residence time of 13 minutes. Both compounds show an AR-selective mode of action with anti-proliferative activity in AR-dependent prostate cancer cell lines (e.g. VCaP, LNCaP, and 22Rv-1) but are inactive against AR-negative prostate cancer cell lines (e.g. DU145). VCaP and mCRPC patient-derived xenograft (PDX) RNA profiling provides evidence of a marked, dose-dependent, pharmacodynamic response indicative of EP300/CBP inhibition. In the VCaP xenograft model, OPN-6602 and OPN-6742 show dose-dependent tumor growth inhibition and corresponding pharmacodynamic responses with prostate-specific antigen level decreases and decreased H3K27 acetylation. In the QR (AR-v7+) PDX model of CRPC (post-androgen deprivation therapy), a synergistic effect was observed between OPN-6602 and enzalutamide. This combination further modulates the expression of AR-driven transcripts such as MYC compared to single agent treatments. The pharmacokinetic profiles of OPN-6602 and OPN-6742 are favorable for oral single daily dose administration due to a high Cmax and short half-life. Due to its higher potency, OPN-6602 has been chosen as clinical candidate and a first-in-human study will start in 2024. Citation Format: Bernice Matusow, Wayne Spevak, Chao Zhang, Yan Ma, Rafe Shellooe, Peipei Li, James Tsai, Pan- Yu Chen, Parmveer Singh, Jackie Walling, Christine Nichols, Jason Halladay, Kerry Inokuchi, Gaston Habets, Gideon Bollag. OPN-6602, a dual EP300/CBP bromodomain inhibitor modulates androgen-driven transcription, including MYC, in mCRPC [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C085.

Discovery of Novel, First-in-Class Allosteric Modulators of MALT1 Scaffolding Function with Differentiated Pharmacology for NFκB-Driven Malignancies

Constitutive activation of NFκB is an oncogenic driver for many lymphoid and selected solid tumor malignancies. BTK inhibitors, which affect the upstream NFκB pathway, have been successfully used in some diseases, but their utility is limited. The majority of DLBCL patients, for instance, are insensitive to BTK inhibition, supporting the need for alternative mechanisms to block NFκB-driven tumor progression. Knockdown of MALT1, a key component of the CBMT complex (CARD-BCL10-MALT1-TRAF6) involved in direct signal transduction of NFκB, is able to block NFκB-dependent DLBCL tumor growth in BTKi sensitive or resistant tumors, but pharmacologic modulation has been challenged by the dual-functionality of MALT1 in a complexed signalosome. MALT1 functions as the scaffolding component of the CBMT complex. Activated MALT1 recruits multiple proteins and directly activates NFκB canonical signaling. MALT1 also has protease activity that fine-tunes various signaling pathways including NFκB via the cleavage of many substrates. The scaffolding activity of MALT1 is critical for cancer cell survival. Most MALT1 inhibitors discovered so far only block protease activity, which results in incomplete NFκB inhibition, suboptimal anti-tumor activity, and the induction of severe autoimmunity due to protease inhibition. Using our Smart Allostery platform, we elucidated and then drugged a natural hotspot on MALT1 that selectively impacts scaffolding but not protease function. The series of scaffolding modulators demonstrate potent inhibition of MALT1 scaffolding activity with cellular IC 50 values ranging from 15 to 250 nM, while exerting minimal influence on MALT1 protease function. Unlike protease inhibitors, which only partially affect NFκB activity, scaffolding modulators completely inhibit the NFκB downstream signaling manifested by the suppression of NFκB activity with cellular IC 50 values ranging from 2 to 90 nM. Consequently, scaffolding modulators exhibit a superior and broader anti-proliferation profile compared to MALT1 protease inhibitors and BTK inhibitors in a panel of lymphoma cell lines. Pathway and genetic analyses indicate that B-cell lymphoma lines driven by NFκB are especially sensitive to scaffolding modulators. In vivo studies demonstrate a robust, dose-dependent tumor growth inhibition (TGI) by one representative scaffolding modulator in several B-cell lymphoma xenograft models. In a protease inhibitor sensitive ABC-DLBCL model, OCI-LY10, our scaffolding modulator achieved a maximal TGI of 94%, while a clinical MALT1 protease inhibitor showed 62% maximal TGI (p<0.001). In protease resistant NFκB driven DLBCL models, MALT1 scaffolding modulation resulted in tumor regression or stasis. These findings highlight the differentiated pharmacology of our scaffolding modulators. Distinct from protease inhibitors that suppress T cell activation and Treg population in vivo, scaffolding modulators also did not deplete Treg cells nor suppress T cell activation in a 14-day study. This potentially mitigates the risk of development of autoimmune disease caused by chronic MALT1 protease inhibition. In summary, we have discovered novel, first-in-class allosteric modulators of MALT1 scaffolding activities. These scaffolding modulators drive enhanced and broader anti-tumor effect in models resistant to MALT1 protease or BTK inhibitors and do so without immune dysregulation. Scaffolding modulation represents a potential best-in-class therapeutic opportunity to modulate MALT1 in patients with NFκB-driven malignancies.

EP102: Pharmacological Inhibition of METTL3 Elicits Tumor Growth Inhibition In Vivo and Demonstrates Synergy with Venetoclax in Various AML Models

METTL3 is the RNA methyltransferase predominantly responsible for the addition of N-6-methyladenosine (m6A), the most abundant modification to mRNA. The prevalence of m6A and the activity and expression of METTL3 have been linked to the appearance and progression of acute myeloid leukemia (AML) 1. EPICS has discovered and optimized a small molecule inhibitor of METTL3 (“M3i”). M3i was shown to inhibit the enzymatic activity of METTL3 (IC 50 = 1.8 nM, SPA assay). The anti-proliferative effects of M3i was demonstrated in various AML cell lines (Kasumi, IC 50 = 63 nM; MV-411, IC 50 = 506 nM; KG1a, IC 50 = 99 nM). Biomarkers were evaluated following M3i treatment of Kasumi and MV-411 cells with consistent results demonstrating depletion of oncoproteins relevant to apoptotic (BCL-2, MCL1) and proliferative (MYC, BRD4, SP1) pathways. In addition, combined treatment with M3i and venetoclax showed clear synergistic effects 2 to inhibit proliferation on both cell lines consistent with the depletion of BCL-2 and MCL1, known targets for venetoclax activity and resistance mechanisms. M3i has been optimized for oral dosing with oral exposure confirmed in mouse, rat and minipig (oral bioavailability >80%, rat; >30%, minipig). In vivo efficacy was evaluated in a disseminated xenograft model using established systemic MV-411-Luc-mCh-Puro 3 in female NSG mice where M3i (10, 30 mg/kg, oral, QDx31 days) demonstrated dose-dependent tumor growth inhibition (p<0.01, relative to vehicle control) of cancer progression as measured by in-life imaging and confirmed by flow cytometry demonstrating significant, dose-dependent pharmacological knockdown of hCD45+ cells in bone marrow, blood and spleen (p<0.01, in all cases). These results confirm that the pharmacological inhibition of METTL3 arrests proliferation of AML and further provide a mechanistic basis for the synergistic activity of M3i in combination with venetoclax as is a viable strategy for the treatment of disease. 1Yankova E et al. (2021), Nature 593:597-601; 2Ianevski A et al. (2022), Nucl Acids Res 50:W739-W743; 3Trachet E (2016), LabCorp Drug Development website: model spotlight MV (4;11)

Preclinical development of 1B7/CD3, a novel anti-TSLPR bispecific antibody that targets CRLF2-rearranged Ph-like B-ALL

Patients harboring CRLF2-rearranged B-lineage acute lymphocytic leukemia (B-ALL) face a 5-year survival rate as low as 20%. While significant gains have been made to position targeted therapies for B-ALL treatment, continued efforts are needed to develop therapeutic options with improved duration of response. Here, first we have demonstrated that patients with CRLF2-rearranged Ph-like ALL harbor elevated thymic stromal lymphopoietin receptor (TSLPR) expression, which is comparable with CD19. Then we present and evaluate the anti-tumor characteristics of 1B7/CD3, a novel CD3-redirecting bispecific antibody (BsAb) that co-targets TSLPR. In vitro, 1B7/CD3 exhibits optimal binding to both human and cynomolgus CD3 and TSLPR. Further, 1B7/CD3 was shown to induce potent T cell activation and tumor lytic activity in both cell lines and primary B-ALL patient samples. Using humanized cell- or patient-derived xenograft models, 1B7/CD3 treatment was shown to trigger dose-dependent tumor remission or growth inhibition across donors as well as induce T cell activation and expansion. Pharmacokinetic studies in murine models revealed 1B7/CD3 to exhibit a prolonged half-life. Finally, toxicology studies using cynomolgus monkeys found that the maximum tolerated dose of 1B7/CD3 was ≤1 mg/kg. Overall, our preclinical data provide the framework for the clinical evaluation of 1B7/CD3 in patients with CRLF2-rearranged B-ALL.

Targeting FAK/PYK2 with SJP1602 for Anti-Tumor Activity in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) presents significant challenges due to its aggressive nature and limited treatment options. Focal adhesion kinase (FAK) has emerged as a critical factor promoting tumor growth and metastasis in TNBC. Despite encouraging results from preclinical and early clinical trials with various FAK inhibitors, none have yet achieved clinical success in TNBC treatment. This study investigates the therapeutic potential of a novel dual inhibitor of FAK and PYK2, named SJP1602, for TNBC. In vitro experiments demonstrate that SJP1602 effectively inhibits FAK and PYK2 activities, showing potent effects on both kinases. SJP1602 shows concentration-dependent inhibition of cell growth, migration, invasion, and 3D spheroid formation in TNBC cell lines, surpassing the efficacy of other FAK inhibitors. Pharmacokinetic studies in rats indicate favorable bioavailability and sustained plasma concentrations of SJP1602, supporting its potential as a therapeutic agent. Furthermore, in TNBC xenograft models, SJP1602 exhibits significant dose-dependent inhibition of tumor growth. These promising results emphasize the potential of SJP1602 as a potent dual inhibitor of FAK and PYK2, deserving further investigation in clinical trials for TNBC treatment.

Discovery of SHR5428 as a selective and noncovalent inhibitor of CDK7

Cyclin dependent kinase 7 (CDK7) is an attractive target in tumor indications via regulating both cell cycle and transcription. Here, SHR5428 was discovered as a selective and noncovalent CDK7 inhibitor with highly potent CDK7 enzymatic activity and triple negative breast cancer cellular activity on MDA-MB-468 cell. SHR5428 also displayed favorable pharmacokinetic properties in different preclinical species such as mouse, rat and dog, and showed high selectivity over CDK1, CDK2, CDK4, CDK6, CDK9, CDK12 in CDK family. Furthermore, the computational modeling has shed some light on this mechanism. Additionally the in vivo efficacy study in a breast cancer cell line (HCC70 cell) derived xenograft mouse model proved SHR5428 to be orally efficacious with dose-dependent tumor growth inhibition.

A targeted thorium-227 conjugate demonstrates efficacy in preclinical models of acquired drug resistance and combination potential with chemotherapeutics and antiangiogenic therapies.

Targeted alpha therapies (TATs) are an innovative class of for cancer treatment. The unique mode-of-action of TATs is the induction of deleterious DNA double-strand breaks. Difficult-to-treat cancers, such as gynecological cancers upregulating the chemoresistance P-glycoprotein (p-gp) and overexpressing the membrane protein mesothelin (MSLN), are promising targets for TATs. Here, based on the previous encouraging findings with monotherapy, we investigated the efficacy of the mesothelin-targeted thorium-227 conjugate (MSLN-TTC) both as monotherapy and in combination with chemotherapies and antiangiogenic compounds in ovarian (OvCa) and cervical cancer models expressing p-gp. MSLN-TTC monotherapy showed equal cytotoxicity in vitro in p-gp-positive and negative cancer cells, while chemotherapeutics dramatically lost activity on p-gp-positive cancer cells. In vivo, MSLN-TTC exhibited dose-dependent tumor growth inhibition with T/C ratios of 0.03-0.44 in various xenograft models irrespective of p-gp expression status. Furthermore, MSLN-TTC was more efficacious in p-gp-expressing tumors than chemotherapeutics. In the MSLN-expressing ST206B OvCa PDX model, MSLN-TTC accumulated specifically in the tumor, which combined with pegylated liposomal doxorubicin (doxil), docetaxel, bevacizumab, or regorafenib treatment induced additive-to-synergistic antitumor efficacy and substantially increased response rates compared with respective monotherapies. The combination treatments were well tolerated and only transient decreases in white and red blood cells were observed. In summary, we demonstrate that MSLN-TTC treatment shows efficacy in p-gp-expressing models of chemoresistance and has combination potential with chemo- and antiangiogenic therapies.

Discovery of N-(2-chloro-5-(3-(pyridin-4-yl)-1H-pyrazolo[3,4-b]pyridin-5-yl)pyridin-3-yl)-4-fluorobenzenesulfonamide (FD274) as a highly potent PI3K/mTOR dual inhibitor for the treatment of acute myeloid leukemia

PI3K-Akt-mTOR pathway is a highly activated signal transduction pathway in human hematological malignancies and has been validated as a promising target for acute myeloid leukemia (AML) therapy. Herein, we designed and synthesized a series of 7-azaindazole derivatives as potent PI3K/mTOR dual inhibitors based on our previously reported FD223. Among them, compound FD274 showed excellent dual PI3K/mTOR inhibitory activity, with IC50 values against PI3Kα/β/γ/δ and mTOR of 0.65 nM, 1.57 nM, 0.65 nM, 0.42 nM, and 2.03 nM, respectively, superior to compound FD223. Compared to the positive drug Dactolisib, FD274 exhibited significant anti-proliferation of AML cell lines (HL-60 and MOLM-16 with IC50 values of 0.092 μM and 0.084 μM, respectively) in vitro. Furthermore, FD274 demonstrated dose-dependent inhibition of tumor growth in the HL-60 xenograft model in vivo, with 91% inhibition of tumor growth at an intraperitoneal injection dose of 10 mg/kg and no observable toxicity. All of these results suggest that FD274 has potential for further development as a promising PI3K/mTOR targeted anti-AML drug candidate.

Discovery of CDK4/6 bifunctional degraders for ER+/HER2- breast cancer.

1083 Background: CDK4/6 inhibitors (CDK4/6i) such as palbociclib and ribociclib are used to treat ER+/HER2- breast cancer, but patients can develop resistance via many mechanisms, several of which converge on the upregulation of CDK6. This has been shown to limit the effectiveness of CDK4/6i in ER+ breast cancer with up to 20% patients exhibiting innate resistance and up to 70% patients developing acquired resistance after 3 years on therapy (Scheidemann, 2021, doi:10.3390/ijms222212292). Methods: To address this limitation, we utilized our PRODEGY platform of Cereblon (CRBN) binders to synthesize CRBN mediated CDK4/6 bifunctional degraders to potently inhibit tumor growth for treatment of naïve ER+/HER2- breast cancer and CDK4/6i resistant tumors. Results: Target degradation by immunoblot analysis of the triple negative breast cancer (TNBC) cell line, MDA-MB-231, treated with our CDK4/6 bifunctional degraders for 6 hours showed up to 85% degradation of CDK4 and CDK6 with DC50s of 1-100nM. CDK4/6 phosphorylates the protein RB which releases the transcription factor E2F, inducing the expression of genes which promote cell cycle progression. Analysis of RB phosphorylation by in-cell western upon 24 hours of CDK4/6 degrader treatment showed phospho-RB IC50s at <30nM. Cell cycle analysis by staining with propidium iodide after 24 hours of treatment with CDK4/6 degraders induced G0/G1 cell cycle arrest at concentrations as low as 10nM. We used a 2D colony formation assay (CFA) as a readout for inhibition of proliferation by cell cycle arrest. Our CDK4/6 degraders showed potent inhibition of cell proliferation with CFA IC50s of <100nM in TNBC cell lines and <25nM in ER+ cell lines, including MCF7, T47D and ZR751 compared to CDK4/6i which ranged from 200nM to 500nM in MCF7 cells. We demonstrated that our CDK4/6 bifunctional degraders were significantly more potent in vitro than the CDK4/6i ribociclib and palbociclib, and the increased activity was due to CRBN mediated target degradation. Our CDK4/6 bifunctional degraders display excellent pharmacokinetic properties in mice with half-lives between 2-10 hours, oral bioavailability between 50-96%. MCF7 xenograft results with our proof-of-concept CDK4/6 degrader showed dose-dependent tumor growth inhibition and greater potency compared to the clinical CDK4/6i ribociclib. We saw tumor regression with our degrader at higher doses which we did not see at any dose of CDK4/6i. Conclusions: Our CDK4/6 bifunctional degraders display excellent single agent activity in vitro and in vivo particularly in comparison to clinically approved CDK4/6i, indicating that using a degrader approach to targeting this pathway may be more effective than current inhibitor therapies.

Old Drug, New Delivery Strategy: MMAE Repackaged.

Targeting therapy is a concept that has gained significant importance in recent years, especially in oncology. The severe dose-limiting side effects of chemotherapy necessitate the development of novel, efficient and tolerable therapy approaches. In this regard, the prostate specific membrane antigene (PSMA) has been well established as a molecular target for diagnosis of, as well as therapy for, prostate cancer. Although most PSMA-targeting ligands are radiopharmaceuticals used in imaging or radioligand therapy, this article evaluates a PSMA-targeting small molecule-drug conjugate, and, thus, addresses a hitherto little-explored field. PSMA binding affinity and cytotoxicity were determined in vitro using cell-based assays. Enzyme-specific cleavage of the active drug was quantified via an enzyme-based assay. Efficacy and tolerability in vivo were assessed using an LNCaP xenograft model. Histopathological characterization of the tumor in terms of apoptotic status and proliferation rate was carried out using caspase-3 and Ki67 staining. The binding affinity of the Monomethyl auristatin E (MMAE) conjugate was moderate, compared to the drug-free PSMA ligand. Cytotoxicity in vitro was in the nanomolar range. Both binding and cytotoxicity were found to be PSMA-specific. Additionally, complete MMAE release could be reached after incubation with cathepsin B. In vivo, the MMAE conjugate displayed good tolerability and dose-dependent inhibition of tumor growth. Immunohistochemical and histological studies revealed the antitumor effect of MMAE.VC.SA.617, resulting in the inhibition of proliferation and the enhancement of apoptosis. The developed MMAE conjugate showed good properties in vitro, as well as in vivo, and should, therefore, be considered a promising candidate for a translational approach.

Abstract 2012: A novel antibody-drug conjugate platform enabling high drug-to-antibody ratios and greater payload flexibility

Abstract Historically, increasing the drug-to-antibody ratio (DAR) in an antibody-drug conjugate (ADC) significantly raises aggregation propensity as a result of the hydrophobicity of the payload. Consequently, DARs are commonly limited to 4, forcing developers to incorporate very high potency payloads to achieve the required efficacy. This, combined with chemical instability and off-target uptake, often results in dose-limiting toxicities and narrow therapeutic indexes. In this study, we used a novel and flexible multivalent linker technology to construct two ADCs targeting HER-2 with the monoclonal antibody, trastuzumab and interchain cysteine bioconjugation. One ADC incorporated monomethyl auristatin E (MMAE) as the drug payload at average DAR 16 whilst the second incorporated the camptothecin analog, SN38, as the drug payload also at average DAR 16. The ADCs were characterized by liquid chromatography-mass spectrometry (LC-MS), size-exclusion chromatography (SEC), hydrophobic interaction chromatography (HIC) and SDS-PAGE and were then tested for efficacy in a range of HER-2 positive cell lines in vitro, and tumor growth inhibition studies using NCI-N87 cells in a mouse xenograft. Both ADCs remained > 95% monomeric (no aggregation) and demonstrated greater chemical stability in ex-vivo serum stability studies compared to comparator ADC constructs utilizing a clinically validated linker and the same payload release chemistry. In in vitro cell killing studies, the ADCs demonstrated target-specific cell killing with the SN38 average DAR 16 ADC achieving a disproportionate 30-fold increase in IC50 (by ADC concentration) compared to a DAR 8 comparator. Dose dependent tumor growth inhibition was observed in an HER-2 positive NCI-N87 mouse xenograft model with no change in mouse body weight, and good systemic exposure. These data demonstrate our capability, using a novel multivalent linker technology, to construct ADCs with DARs considerably greater than DAR 8 which are chemically stable, shield payloads from hydrophobic interactions (no aggregation), demonstrate good in vivo exposure profiles and which are highly efficacious in vitro and in vivo. Higher DARs will permit the incorporation of lower potency and targeted small molecule payloads into ADCs which will result in targeted cancer therapeutics with wider therapeutic indexes through the delivery of greater concentrations of payload to the tumor microenvironment and lower, payload-associated, dose-limiting toxicity. Citation Format: Ludovic Juen, Adam J. Collier, Anthony W. Tolcher, Myriam M. Ouberai. A novel antibody-drug conjugate platform enabling high drug-to-antibody ratios and greater payload flexibility [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2012.

Abstract 1618: A highly selective and potent TLR8 agonist ZG0895 in preclinical studies of anti-tumor activities

Abstract Toll-like receptor 8 (TLR8) recognizes ssRNA fragments from pathogens and initiate both innate and adaptive immune responses. TLR8 activation can reverse Treg and myeloid-derived suppressor cells (MDSC) mediated immune suppression by tumors that lead to a restore of immune response to inhibit tumor growth. ZG0895 is a potent and novel TLR agonist that shows a highly selective activation of TLR8 and has a more than 300-fold potency over TLR7 in EC50 values. The agonist screening results further confirmed that ZG0895 without activation of TLR isoforms like TLR2, 4 or 9 in cells. In pharmacokinetics and pharmacodynamics, ZG0895 has a preferable ADME and PK profile and can activate immune system via subcutaneous (s.c.) administration. For in vivo anti-tumor efficacy studies, ZG0895 at 1~30 mg/kg exhibited significant dose-dependent inhibition of tumor growth in immunocompetent myeloid-sufficient murine models with CT26, EMT6 or HH subcutaneous xenografts. Furthermore, ZG0895 administration resulted in a complete tumor regression by ZG0895 at high dose via s.c. injection. Mechanism studies of ZG0895 in ex vivo human PBMC assay showed a strong induction of TLR8 related cytokines. In vivo study in tumor-bearing mouse demonstrated that pro-inflammatory cytokines, such as TNFα, IL12p40, IL6 and IL10, were quickly released after ZG0895 treatment but soon returned to normal that can avoid continuous activation of systemic immunity and reduce the risk of cytokine release storm. Consistently, in vivo cynomolgus monkey study by consecutive treatments with ZG0895 (day 30) resulted in a significant increase of immune system cells including intermediate monocytes, non-classic monocytes, cDCs and NK cells, along with notable decrease of classical monocytes but unchanged pDCs, suggesting a possible reversion of tumor-initiated MDSC via TLR8 activation. All these changes were back to normal after period of recovery (day 57), indicated the immune effect of ZG0895 was restrained. These preclinical results support that ZG0895 as a highly TLR8-selective agonist inhibits tumor growth via the activation of TLR8 eliciting immune responses but under a better safety than other conventional TLR8 agonists, and it is worth to be further explored as an anti-cancer drug candidate. Citation Format: Dawei Cui, Ruifeng Liu, Huanyi Yang, Bing Zhu, Zelin Sheng, Binhua Lv. A highly selective and potent TLR8 agonist ZG0895 in preclinical studies of anti-tumor activities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1618.

Abstract 2790: Preclinical studies of ZG19018, a novel irreversible covalent inhibitor of KRAS G12C, for the treatment of advanced non-small cell lung cancer and other solid tumors

Abstract Growing evidences of covalent irreversible inhibitors in clinical treatments of solid tumors with KRAS G12C mutation have recently emerged after FDA-approved AMG510 (Sotorasib) for NSCLC chemotherapies. ZG19018 is an irreversible KRAS G12C inhibitor with novel structure that shows promising preclinical results for in vitro molecular and biological studies and in vivo G12C-mutated cancer models and safety studies in animals. For instance, ZG19018 rapidly and selectively forms covalent bonds with G12C-GDP, which accounts for lock-in KRAS protein as the inactive GDP-bound state but inhibits the activation of numerous downstream signaling pathways like RAF/MEK on ERK phosphorylation. ZG19018 showed highly inhibition of a 3D growth panel for human cancer cell lines harboring the KRAS G12C mutation, e.g. H358 (NSCLCs), MIA PaCa-2 (pancreatic) and SW837 (colorectal), etc. For pharmacodynamics, a battery of in vitro and in vivo ADME/DMPK studies indicated a high plasma protein binding (>99.6%) of ZG19018 with over 40% bioavailability in rat and dog via oral administration for the adequate drug levels in various tissues including lung, crossing BBB to reach brain, and accumulation in tumors. For in vivo anti-tumor efficacy, ZG19018 exhibited significant dose-dependent inhibition of tumor growth of G12C-driven subcutaneous models in nude mouse like H358 NSCLC cells but induced further tumor regression in pancreatic cancer MIA PaCa-2 xenograft. ZG19018 also showed an enhanced anti-tumor activities as combined with chemo-reagent Donafinib, a broad tyrosine kinase inhibitor blocking RAF/MEK/ERK signaling pathways of tumor cells, in the same H358 xenograft model. The general toxicology studies in rat under 28 days repeated oral administration (q.d.) showed NOAEL and STD10 values at 75 and 150 mg/kg/day, respectively. The safety pharmacological studies showed no notable effects of ZG19018 on the cardiovascular, respiratory and CNS systems. The genotoxic potential studies indicated ZG19018 without mutagenesis. Collectively, ZG19018 is now in phase I/II clinical trials for the treatments of solid tumors with KRAS G12C mutation in China. Citation Format: Bing Zhu, Dawei Cui, Ruifeng Liu, Weidong Feng, Zelin Sheng, Binhua Lv. Preclinical studies of ZG19018, a novel irreversible covalent inhibitor of KRAS G12C, for the treatment of advanced non-small cell lung cancer and other solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2790.

Abstract 517: Targeting chromosomally unstable tumors with a selective KIF18A inhibitor

Abstract Chromosome instability (CIN) is an attribute of cancer cells that has yet to be fully exploited therapeutically, despite its prevalence across many tumor types and lack of occurrence in normal cells. To identify genes uniquely essential to CINhigh cells, we mined the Cancer Dependency map (DepMap) for genes required for proliferation of tumor cell lines with high levels of copy number aberrations, an indicator of CIN. This analysis identified KIF18A, a mitotic kinesin also identified by others as a vulnerability of aneuploid and whole genome doubled cells. Genetic disruption of KIF18A with RNAi confirmed its necessity in CINhigh cells, and these findings translated to in vivo reduction in tumor growth, illustrating the potential of KIF18A as a therapeutic target. High-throughput screening of a chemical library for inhibitors of KIF18A motor domain’s ATPase activity identified an ATP-noncompetitive hit that was optimized for potency, selectivity, and pharmacokinetic properties. These optimized inhibitors are orally bioavailable, exhibit sub-nM potency, and are highly selective for KIF18A vs. other kinesins. They also display a long drug-target residence time, a unique property that allows cells to be trapped in mitosis long enough to result in cell death. Mechanistically, inhibition of KIF18A’s ATPase activity renders KIF18A unable to localize to the metaphase plate, instead becoming trapped at the spindle poles. This inability to translocate results in defects in chromosome congression, evidenced by a less compact metaphase plate, and an accumulation of cells in G2/M. Treatment of CIN cancer cells with these inhibitors results in a proliferative defect due in part to induction of programmed cell death. In contrast to the effect in cancer cells, KIF18A inhibition has no effect on rapidly proliferating non-transformed epithelial cells and primary T cells. The differential impact between normal and cancer cells sets KIF18A inhibitors apart from other anti-mitotic therapies, which have had limited clinical benefit due to their detrimental impact on both cancer and highly proliferative normal cells. The therapeutic potential of KIF18A inhibitors was assessed in human tumor xenograft models. Treatment of multiple tumor models resulted in substantial, dose-dependent inhibition of tumor growth. KIF18A inhibition in the tumor was confirmed through evaluation of KIF18A localization changes, chromosome congression and an increase in mitotic index. The strong biological rationale, robust preclinical data, and superior compound properties warrant clinical development of Volastra’s KIF18A inhibitor which is planned for development for the treatment of chromosomally unstable cancers. Citation Format: Aaron F. Phillips, Rumin Zhang, Akanksha Verma, Tamar Feinberg, Mia Jaffe, Marysol Chu Carty, Ryan Schulz, Sarah Bettigole, Celia Andreu, Scott Drutman, Michael Su, Derek Cogan, Christina H. Eng. Targeting chromosomally unstable tumors with a selective KIF18A inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 517.

Abstract 3451: XS-04: A first-in-class dual IRAK4/BTK inhibitor for the treatment of hematological malignancies

Abstract Bruton's tyrosine kinase (BTK), a critical mediator of B-cell receptor (BCR) signaling, is a validated therapeutic target found across multiple B-cell cancers. Though initially responding to treatment with a BTK inhibitor, many patients with chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), or Waldenström macroglobulinemia (WM) eventually progressed and clinical responses to BTK inhibitors are often poor in diffuse large B-cell lymphoma (DLBCL) patients. These highlight the need to understand the resistance mechanisms and improve responses to BTK inhibitors. The serine/threonine protein kinase IRAK4 is a key modulator of MYD88, which is found to be mutated in various aggressive B-cell lymphomas, including DLBCL. As BCR signaling and MYD88 signaling converge downstream on NF-kB activation, which plays an essential role in proliferation and survival of lymphoma cells, blockade of the two signaling pathways presents a potential therapeutic strategy. Here, we investigated the pharmacokinetic properties and anti-tumor activity of XS-04, a first-in-class dual inhibitor targeting both IRAK4 and BTK, using preclinical models of DLBCL subtypes, including tumors resistant to BTK inhibitors. XS-04 showed potent inhibition of IRAK4 and BTK kinases, with IC50 values of 0.2 and 2.9 nM, respectively. Favorable pharmacokinetic properties were also seen across multiple non-clinical species. Using in vitro anti-proliferative assays, we found that XS-04 inhibited proliferation of various DLBCL cell lines with IC50s of 30 to 40 nM. Mechanistically, XS-04 suppressed NF-kB activity in DLBCL cells and in vivo xenograft tumors, as indicated by decreased phosphorylation of IkBa. Oral dosing of XS-04 in TMD8 xenograft model led to a dose-dependent inhibition of tumor growth, which was comparable to the inhibition shown by a combination of BTK and IRAK4 inhibitors. In addition, XS-04 treatment resulted in tumor regression in a patient-derived ibrutinib-resistant MCL xenograft model, without significant changes in body weight. XS-04, a first-in-class IRAK4/BTK dual inhibitor, shows broad anti-tumor activity across preclinical models of DLBCL subtypes, including tumors resistant to BTK inhibitors. These results support XS-04 as a therapeutic candidate for the treatment of hematological malignancies. An IND submission is planned for 2023. Citation Format: Chunyan Zhao, Jianfei Wang, Aiguo Liu, Xin Gao, Yanfen Teng, Lei Liu, Yang Zhang, Xiaohong Yu. XS-04: A first-in-class dual IRAK4/BTK inhibitor for the treatment of hematological malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3451.

Abstract 5045: NTSR1-targeted alpha therapeutic [Ac-225]-FPI-2059 induces growth inhibition in a preclinical colorectal tumor model

Abstract Background: Neurotensin receptor 1 (NTSR1) is overexpressed in multiple cancer indications that include pancreatic, colorectal and prostate cancers, all of which have limited therapeutic treatment options and unmet medical need. Fusion is developing novel targeted alpha therapeutics (TATs) that enable the specific delivery of high energy alpha particles (actinium-225; [225Ac]) to tumor cells while sparing surrounding normal tissues. The alpha radiation released by TATs causes cell damage through the induction of multiple double-stranded DNA breaks leading to tumor cell death. Here, we describe the therapeutic efficacy of an [225Ac]-conjugated, NTSR1 targeting small molecule in a colorectal cancer tumor model. Materials and Methods: CT26 colorectal cancer cells overexpressing murine NTSR1 (mNTSR1) were generated by lentiviral transduction. Selected cells were evaluated for stable mNTSR1 expression by an in vitro radioligand binding assay and subsequently implanted subcutaneously into Balb/c mice for in vivo evaluations. FPI-2056 (parent compound) was radiolabeled with either lutetium-177 ([177Lu]-FPI-2057) or actinium-225 ([225Ac]-FPI-2059). Biodistribution assessment studies were conducted in mice bearing CT26-mNTSR1 tumors dosed intravenously with [177Lu]-FPI-2057. Therapeutic efficacy studies were conducted by intravenous administration of single doses of 0.185 - 5.55 MBq/kg of [225Ac]-FPI-2059 (0.1-3 µCi) to animals bearing CT26-mNTSR1 tumors, followed by tumor growth monitoring for 50 days. Study endpoints included tumor volume measurements and impact on animal health status. Results: Evaluation of [177Lu]-FPI-2057 biodistribution and excretion revealed rapid renal clearance via urine with a clearance from the blood by 24 h. Uptake of [177Lu]-FPI-2057 was detected in the CT26-mNTSR1 tumors with a maximum concentration of 7.0 %ID/g at 6 h post-injection, dropping to 4.6 and 2.8 %ID/g at 24 and 48 h post-injection, with 20-fold higher uptake in the tumor vs. blood levels at both 24 and 48h time points. Therapeutic administration of a single dose of [225Ac]-FPI-2059 resulted in dose-dependent tumor growth inhibition at doses above 1.85 MBq/kg of [225Ac]-FPI-2059 (1 μCi), which translated into increased survival compared to control animals. Conclusion: These results demonstrate that targeted delivery of [225Ac]-FPI-2059 to NTSR1 expressing tumors results in significant growth inhibition and enhanced survival, thereby providing promising preclinical evidence to support the clinical development of [225Ac]-FPI-2059. Citation Format: Saleemulla Mahammad, Jaline Broqueza, Brigitte L. Theriault, John Forbes, Christopher P. Leamon, John Valliant. NTSR1-targeted alpha therapeutic [Ac-225]-FPI-2059 induces growth inhibition in a preclinical colorectal tumor model. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5045.