Discovery of YAP1/TAZ pathway inhibitors through phenotypic screening with potent anti-tumor activity via blockade of Rho-GTPase signaling.

This study describes the identification and target deconvolution of small molecule inhibitors of oncogenic Yes-associated protein (YAP1)/TAZ activity with potent anti-tumor activity in vivo. A high-throughput screen (HTS) of 3.8 million compounds was conducted using a cellular YAP1/TAZ reporter assay. Target deconvolution studies identified the geranylgeranyltransferase-I (GGTase-I) complex as the direct target of YAP1/TAZ pathway inhibitors. The small molecule inhibitors block the activation of Rho-GTPases, leading to subsequent inactivation of YAP1/TAZ and inhibition of cancer cell proliferation in vitro. Multi-parameter optimization resulted in BAY-593, an in vivo probe with favorable PK properties, which demonstrated anti-tumor activity and blockade of YAP1/TAZ signaling in vivo.

Dual targeting of the androgen receptor and PI3K/AKT/mTOR pathways in prostate cancer models improves antitumor efficacy and promotes cell apoptosis.

Prostate cancer is a frequent malignancy in older men and has a very high 5-year survival rate if diagnosed early. The prognosis is much less promising if the tumor has already spread outside the prostate gland. Targeted treatments mainly aim at blocking androgen receptor (AR) signaling and initially show good efficacy. However, tumor progression due to AR-dependent and AR-independent mechanisms is often observed after some time, and novel treatment strategies are urgently needed. Dysregulation of the PI3K/AKT/mTOR pathway in advanced prostate cancer and its implication in treatment resistance has been reported. We compared the impact of PI3K/AKT/mTOR pathway inhibitors with different selectivity profiles on in vitro cell proliferation and on caspase 3/7 activation as a marker for apoptosis induction, and observed the strongest effects in the androgen-sensitive prostate cancer cell lines VCaP and LNCaP. Combination treatment with the AR inhibitor darolutamide led to enhanced apoptosis in these cell lines, the effects being most pronounced upon cotreatment with the pan-PI3K inhibitor copanlisib. A subsequent transcriptomic analysis performed in VCaP cells revealed that combining darolutamide with copanlisib impacted gene expression much more than individual treatment. A comprehensive reversal of the androgen response and the mTORC1 transcriptional programs as well as a marked induction of DNA damage was observed. Next, an in vivo efficacy study was performed using the androgen-sensitive patient-derived prostate cancer (PDX) model LuCaP 35 and a superior efficacy was observed after the combined treatment with copanlisib and darolutamide. Importantly, immunohistochemistry analysis of these treated tumors showed increased apoptosis, as revealed by elevated levels of cleaved caspase 3 and Bcl-2-binding component 3 (BBC3). In conclusion, these data demonstrate that concurrent blockade of the PI3K/AKT/mTOR and AR pathways has superior antitumor efficacy and induces apoptosis in androgen-sensitive prostate cancer cell lines and PDX models.

Mono- and multimeric PSMA-targeting small molecule-thorium-227 conjugates for optimized efficacy and biodistribution in preclinical models.

PURPOSE: PSMA (prostate-specific membrane antigen) is highly expressed on prostate cancer (PrCa) cells and extensively used as a homing target for PrCa treatment. Most prominently, PSMA-targeting conjugate PSMA-617, carrying a DOTA chelator and labeled with therapeutic radionuclides like beta-emitting lutetium-177 or alpha-emitting actinium-225, has shown clinical activity in PrCa patients. We sought to develop PSMA-targeting small molecule (SMOL) conjugates that show high uptake in PSMA-expressing tumors and fast clearance, and can easily be labeled with the alpha emitter thorium-227 (half-life 18.7 days). METHODS: A novel linker motif with improved competition against 3H-PSMA-617 on PSMA-expressing LNCaP cells was identified. A 2,3-hydroxypyridinone chelator modified with carboxyl groups (carboxy-HOPO) with increased hydrophilicity and robust labeling with thorium-227 was developed and allowed the synthesis of mono-, di-, tri-, and tetrameric conjugates. The resulting monomeric and multimeric PSMA SMOL-TTCs (targeted thorium conjugate) were evaluated for cellular binding, internalization, and antiproliferative activity. The in vivo antitumor efficacy of the PSMA SMOL-TTCs was determined in ST1273 and KUCaP-1 PrCa models in mice, and their biodistribution was assessed in cynomolgus monkeys, minipigs, and mice. RESULTS: The monomeric and multimeric PSMA SMOL conjugates were readily labeled with thorium-227 at room temperature and possessed high stability and good binding, internalization, and antiproliferative activity in vitro. In vivo, the monomeric, dimeric, and trimeric PSMA SMOL-TTCs showed fast clearance, potent antitumor efficacy, and high uptake and retention in prostate tumors in mice. No major uptake or retention in other organs was observed beyond kidneys. Low uptake of free thorium-227 into bone confirmed high complex stability in vivo. Salivary gland uptake remained inconclusive as mini pigs were devalidated as a relevant model and imaging controls failed in cynomolgus monkeys. CONCLUSION: Monomeric and multimeric PSMA SMOL-TTCs show high tumor uptake and fast clearance in preclinical models and warrant further therapeutic exploration.

Zoledronic Acid Prevents Bone Resorption Caused by the Combination of Radium-223, Abiraterone Acetate, and Prednisone in an Intratibial Prostate Cancer Mouse Model.

An increased risk of non-pathological fractures in patients with prostate cancer and bone metastases has been associated with combination treatment with radium-223, abiraterone, and prednisone/prednisolone in the absence of bone-protecting agents. Here, we investigated possible mechanisms leading to this outcome using an intratibial LNCaP model mimicking prostate cancer bone metastases. Male NOD.scid mice were inoculated intratibially with LNCaP prostate cancer cells and treated with vehicle, radium-223, abiraterone, prednisone, zoledronic acid, or their combinations for 28 days. Serum TRACP 5b and PSA levels were measured. Bone structure, quality, and formation rate of non-tumor-bearing and tumor-bearing tibiae were analyzed by microCT, 3-point bending assay, and dynamic histomorphometry, respectively. Radium-223 incorporation into bone was also measured. Radium-223/abiraterone/prednisone combination treatment induced a transient increase in bone resorption indicated by elevated TRACP 5b levels, which was inhibited by concurrent treatment with zoledronic acid. Furthermore, radium-223/abiraterone/prednisone combination reduced periosteal and trabecular new bone formation and the number of osteoblasts, but bone structure or biomechanical quality were not affected. The abiraterone/prednisone treatment decreased radium-223 incorporation into tumor-bearing bone, possibly explaining the lack of additional antitumor efficacy. In conclusion, radium-223/abiraterone/prednisone combination increased bone resorption, which may have been one of the mechanisms leading to an increased fracture risk in patients with mCRPC.

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 (TAT) are an innovative class of therapies 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 gynecologic 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 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 treatment/control 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 ovarian cancer patient-derived xenograft 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.

Enhanced Antitumor Efficacy of Radium-223 and Enzalutamide in the Intratibial LNCaP Prostate Cancer Model.

Radium-223 dichloride and enzalutamide are indicated for metastatic castration-resistant prostate cancer and their combination is currently being investigated in a large phase 3 clinical trial. Here, we evaluated the antitumor efficacy of radium-223, enzalutamide, and their combination in the intratibial LNCaP model mimicking prostate cancer metastasized to bone. In vitro experiments revealed that the combination of radium-223 and enzalutamide inhibited LNCaP cell proliferation and showed synergistic efficacy. The combination of radium-223 and enzalutamide also demonstrated enhanced in vivo antitumor efficacy, as determined by measuring serum PSA levels in the intratibial LNCaP model. A decreasing trend in the total area of tumor-induced abnormal bone was associated with the combination treatment. The serum levels of the bone formation marker PINP and the bone resorption marker CTX-I were lowest in the combination treatment group and markedly decreased compared with vehicle group. Concurrent administration of enzalutamide did not impair radium-223 uptake in tumor-bearing bone or the ability of radium-223 to inhibit tumor-induced abnormal bone formation. In conclusion, combination treatment with radium-223 and enzalutamide demonstrated enhanced antitumor efficacy without compromising the integrity of healthy bone. The results support the ongoing phase 3 trial of this combination.

A Novel NAMPT Inhibitor-Based Antibody-Drug Conjugate Payload Class for Cancer Therapy.

Inhibition of intracellular nicotinamide phosphoribosyltransferase (NAMPT) represents a new mode of action for cancer-targeting antibody-drug conjugates (ADCs) with activity also in slowly proliferating cells. To extend the repertoire of available effector chemistries, we have developed a novel structural class of NAMPT inhibitors as ADC payloads. A structure-activity relationship-driven approach supported by protein structural information was pursued to identify a suitable attachment point for the linker to connect the NAMPT inhibitor with the antibody. Optimization of scaffolds and linker structures led to highly potent effector chemistries which were conjugated to antibodies targeting C4.4a (LYPD3), HER2 (c-erbB2), or B7H3 (CD276) and tested on antigen-positive and -negative cancer cell lines. Pharmacokinetic studies, including metabolite profiling, were performed to optimize the stability and selectivity of the ADCs and to evaluate potential bystander effects. Optimized NAMPTi-ADCs demonstrated potent in vivo antitumor efficacy in target antigen-expressing xenograft mouse models. This led to the development of highly potent NAMPT inhibitor ADCs with a very good selectivity profile compared with the corresponding isotype control ADCs. Moreover, we demonstrate─to our knowledge for the first time─the generation of NAMPTi payload metabolites from the NAMPTi-ADCs in vitro and in vivo. In conclusion, NAMPTi-ADCs represent an attractive new payload class designed for use in ADCs for the treatment of solid and hematological cancers.

Identification of a neural development gene expression signature in colon cancer stem cells reveals a role for EGR2 in tumorigenesis.

Recent evidence demonstrates that colon cancer stem cells (CSCs) can generate neurons that synapse with tumor innervating fibers required for tumorigenesis and disease progression. Greater understanding of the mechanisms that regulate CSC driven tumor neurogenesis may therefore lead to more effective treatments. RNA-sequencing analyses of ALDHPositive CSCs from colon cancer patient-derived organoids (PDOs) and xenografts (PDXs) showed CSCs to be enriched for neural development genes. Functional analyses of genes differentially expressed in CSCs from PDO and PDX models demonstrated the neural crest stem cell (NCSC) regulator EGR2 to be required for tumor growth and to control expression of homebox superfamily embryonic master transcriptional regulator HOX genes and the neural stem cell and master cell fate regulator SOX2. These data support CSCs as the source of tumor neurogenesis and suggest that targeting EGR2 may provide a therapeutic differentiation strategy to eliminate CSCs and block nervous system driven disease progression.

Antibody-drug conjugates harboring a kinesin spindle protein inhibitor with immunostimulatory properties.

Antibody-drug conjugates (ADCs) are used to target cancer cells by means of antibodies directed to tumor-associated antigens, causing the incorporation of a cytotoxic payload into target cells. Here, we characterized the mode of action of ADC costing of a TWEAKR-specific monoclonal antibody conjugated to a small molecule kinesin spindle protein (KSP) inhibitor (KSPi). These TWEAKR-KSPi-ADCs showed strong efficacy in a TWEAKR expressing CT26 colon cancer model in mice. TWEAKR-KSPi-ADCs controlled the growth of CT26 colon cancers in immunodeficient as well as in immunocompetent mice. However, when treated with suboptimal doses, TWEAKR-KSPi-ADCs were still active in immunocompetent but not in immunodeficient mice, indicating that TWEAKR-KSPi-ADCs act - in addition to the cytotoxic mode of action - through an immunological mechanism. Indeed, in vitro experiments performed with a cell-permeable small molecule KSPi closely related to the active payload released from the TWEAKR-KSPi-ADCs revealed that KSPi was capable of stimulating several hallmarks of immunogenic cell death (ICD) on three different human cancer cell lines: cellular release of adenosine triphosphate (ATP) and high mobility group B1 protein (HMGB1), exposure of calreticulin on the cell surface as well as a transcriptional type-I interferon response. Further, in vivo experiments confirmed that treatment with TWEAKR-KSPi-ADCs activated immune responses via enhancing the infiltration of CD4+ and CD8+ T lymphocytes in tumors and the local production of interferon-γ, interleukin-2, and tumor necrosis factor-α. In conclusion, the antineoplastic effects of TWEAKR-KSPi-ADCs can partly be attributed to its ICD-stimulatory properties.

Comparative Proteomic and Transcriptomic Analysis of the Impact of Androgen Stimulation and Darolutamide Inhibition.

Several inhibitors of androgen receptor (AR) function are approved for prostate cancer treatment, and their impact on gene transcription has been described. However, the ensuing effects at the protein level are far less well understood. We focused on the AR signaling inhibitor darolutamide and confirmed its strong AR binding and antagonistic activity using the high throughput cellular thermal shift assay (CETSA HT). Then, we generated comprehensive, quantitative proteomic data from the androgen-sensitive prostate cancer cell line VCaP and compared them to transcriptomic data. Following treatment with the synthetic androgen R1881 and darolutamide, global mass spectrometry-based proteomics and label-free quantification were performed. We found a generally good agreement between proteomic and transcriptomic data upon androgen stimulation and darolutamide inhibition. Similar effects were found both for the detected expressed genes and their protein products as well as for the corresponding biological programs. However, in a few instances there was a discrepancy in the magnitude of changes induced on gene expression levels compared to the corresponding protein levels, indicating post-transcriptional regulation of protein abundance. Chromatin immunoprecipitation DNA sequencing (ChIP-seq) and Hi-C chromatin immunoprecipitation (HiChIP) revealed the presence of androgen-activated AR-binding regions and long-distance AR-mediated loops at these genes.

DNA repair inhibitors sensitize cells differently to high and low LET radiation.

The aim of this study was to investigate effects of high LET α-radiation in combination with inhibitors of DDR (DNA-PK and ATM) and to compare the effect with the radiosensitizing effect of low LET X-ray radiation. The various cell lines were irradiated with α-radiation and with X-ray. Clonogenic survival, the formation of micronuclei and cell cycle distribution were studied after combining of radiation with DDR inhibitors. The inhibitors sensitized different cancer cell lines to radiation. DNA-PKi affected survival rates in combination with α-radiation in selected cell lines. The sensitization enhancement ratios were in the range of 1.6-1.85 in cancer cells. ATMi sensitized H460 cells and significantly increased the micronucleus frequency for both radiation qualities. ATMi in combination with α-radiation reduced survival of HEK293. A significantly elicited cell cycle arrest in G2/M phase after co-treatment of ATMi with α-radiation and X-ray. The most prominent treatment effect was observed in the HEK293 by combining α-radiation and inhibitions. ATMi preferentially sensitized cancer cells and normal HEK293 cells to α-radiation. DNA-PKi and ATMi can sensitize cancer cells to X-ray, but the effectiveness was dependent on cancer cells itself. α-radiation reduced proliferation in primary fibroblast without G2/M arrest.

Discovery and Characterization of the Potent and Highly Selective 1,7-Naphthyridine-Based Inhibitors BAY-091 and BAY-297 of the Kinase PIP4K2A.

PIP4K2A is an insufficiently studied type II lipid kinase that catalyzes the conversion of phosphatidylinositol-5-phosphate (PI5P) into phosphatidylinositol 4,5-bisphosphate (PI4,5P2). The involvement of PIP4K2A/B in cancer has been suggested, particularly in the context of p53 mutant/null tumors. PIP4K2A/B depletion has been shown to induce tumor growth inhibition, possibly due to hyperactivation of AKT and reactive oxygen species-mediated apoptosis. Herein, we report the identification of the novel potent and highly selective inhibitors BAY-091 and BAY-297 of the kinase PIP4K2A by high-throughput screening and subsequent structure-based optimization. Cellular target engagement of BAY-091 and BAY-297 was demonstrated using cellular thermal shift assay technology. However, inhibition of PIP4K2A with BAY-091 or BAY-297 did not translate into the hypothesized mode of action and antiproliferative activity in p53-deficient tumor cells. Therefore, BAY-091 and BAY-297 serve as valuable chemical probes to study PIP4K2A signaling and its involvement in pathophysiological conditions such as cancer.

Immunostimulatory effects of targeted thorium-227 conjugates as single agent and in combination with anti-PD-L1 therapy.

BACKGROUND: Targeted thorium-227 conjugates (TTCs) are an emerging class of targeted alpha therapies (TATs). Their unique mode of action (MoA) is the induction of difficult-to-repair clustered DNA double-strand breaks. However, thus far, their effects on the immune system are largely unknown. Here, we investigated the immunostimulatory effects of the mesothelin-targeted thorium-227 conjugate (MSLN-TTC) in vitro and in vivo in monotherapy and in combination with an inhibitor of the immune checkpoint programmed death receptor ligand 1 (PD-L1) in immunocompetent mice. METHODS: The murine cell line MC38 was transfected with the human gene encoding for MSLN (hMSLN) to enable binding of the non-cross-reactive MSLN-TTC. The immunostimulatory effects of MSLN-TTC were studied in vitro on human cancer cell lines and MC38-hMSLN cells. The efficacy and MoA of MSLN-TTC were studied in vivo as monotherapy or in combination with anti-PD-L1 in MC38-hMSLN tumor-bearing immunocompetent C57BL/6 mice. Experiments were supported by RNA sequencing, flow cytometry, immunohistochemistry, mesoscale, and TaqMan PCR analyses to study the underlying immunostimulatory effects. In vivo depletion of CD8+ T cells and studies with Rag2/Il2Rg double knockout C57BL/6 mice were conducted to investigate the importance of immune cells to the efficacy of MSLN-TTC. RESULTS: MSLN-TTC treatment induced upregulation of DNA sensing pathway transcripts (IL-6, CCL20, CXCL10, and stimulator of interferon genes (STING)-related genes) in vitro as determined by RNASeq analysis. The results, including phospho-STING activation, were confirmed on the protein level. Danger-associated molecular pattern molecules were upregulated in parallel, leading to dendritic cell (DC) activation in vitro. MSLN-TTC showed strong antitumor activity (T:C 0.38, p<0.05) as a single agent in human MSLN-expressing MC38 tumor-bearing immunocompetent mice. Combining MSLN-TTC with anti-PD-L1 further enhanced the efficacy (T:C 0.08, p<0.001) as evidenced by the increased number of tumor-free surviving animals. MSLN-TTC monotherapy caused migration of CD103+ cDC1 DCs and infiltration of CD8+ T cells into tumors, which was enhanced on combination with anti-PD-L1. Intriguingly, CD8+ T-cell depletion decreased antitumor efficacy. CONCLUSIONS: These in vitro and in vivo data on MSLN-TTC demonstrate that the MoA of TTCs involves activation of the immune system. The findings are of relevance for other targeted radiotherapies and may guide clinical combination strategies.

BAY-8400: A Novel Potent and Selective DNA-PK Inhibitor which Shows Synergistic Efficacy in Combination with Targeted Alpha Therapies.

Eukaryotes have evolved two major pathways to repair potentially lethal DNA double-strand breaks. Homologous recombination represents a precise, DNA-template-based mechanism available during the S and G2 cell cycle phase, whereas non-homologous end joining, which requires DNA-dependent protein kinase (DNA-PK), allows for fast, cell cycle-independent but less accurate DNA repair. Here, we report the discovery of BAY-8400, a novel selective inhibitor of DNA-PK. Starting from a triazoloquinoxaline, which had been identified as a hit from a screen for ataxia telangiectasia and Rad3-related protein (ATR) inhibitors with inhibitory activity against ATR, ATM, and DNA-PK, lead optimization efforts focusing on potency and selectivity led to the discovery of BAY-8400. In in vitro studies, BAY-8400 showed synergistic activity of DNA-PK inhibition with DNA damage-inducing targeted alpha therapy. Combination of PSMA-targeted thorium-227 conjugate BAY 2315497 treatment of human prostate tumor-bearing mice with BAY-8400 oral treatment increased antitumor efficacy, as compared to PSMA-targeted thorium-227 conjugate monotherapy.

Changing for the Better: Discovery of the Highly Potent and Selective CDK9 Inhibitor VIP152 Suitable for Once Weekly Intravenous Dosing for the Treatment of Cancer.

Selective inhibition of exclusively transcription-regulating positive transcription elongation factor b/CDK9 is a promising new approach in cancer therapy. Starting from atuveciclib, the first selective CDK9 inhibitor to enter clinical development, lead optimization efforts aimed at identifying intravenously (iv) applicable CDK9 inhibitors with an improved therapeutic index led to the discovery of the highly potent and selective clinical candidate VIP152. The evaluation of various scaffold hops was instrumental in the identification of VIP152, which is characterized by the underexplored benzyl sulfoximine group. VIP152 exhibited the best preclinical overall profile in vitro and in vivo, including high efficacy and good tolerability in xenograft models in mice and rats upon once weekly iv administration. VIP152 has entered clinical trials for the treatment of cancer with promising longterm, durable monotherapy activity in double-hit diffuse large B-cell lymphoma patients.

Degradation of CCNK/CDK12 is a druggable vulnerability of colorectal cancer.

Novel treatment options for metastatic colorectal cancer (CRC) are urgently needed to improve patient outcome. Here, we screen a library of non-characterized small molecules against a heterogeneous collection of patient-derived CRC spheroids. By prioritizing compounds with inhibitory activity in a subset of-but not all-spheroid cultures, NCT02 is identified as a candidate with minimal risk of non-specific toxicity. Mechanistically, we show that NCT02 acts as molecular glue that induces ubiquitination of cyclin K (CCNK) and proteasomal degradation of CCNK and its complex partner CDK12. Knockout of CCNK or CDK12 decreases proliferation of CRC cells in vitro and tumor growth in vivo. Interestingly, sensitivity to pharmacological CCNK/CDK12 degradation is associated with TP53 deficiency and consensus molecular subtype 4 in vitro and in patient-derived xenografts. We thus demonstrate the efficacy of targeted CCNK/CDK12 degradation for a CRC subset, highlighting the potential of drug-induced proteolysis for difficult-to-treat types of cancer.

RNA sequencing of long-term label-retaining colon cancer stem cells identifies novel regulators of quiescence.

Recent data suggest that therapy-resistant quiescent cancer stem cells (qCSCs) are the source of relapse in colon cancer. Here, using colon cancer patient-derived organoids and xenografts, we identify rare long-term label-retaining qCSCs that can re-enter the cell cycle to generate new tumors. RNA sequencing analyses demonstrated that these cells display the molecular hallmarks of quiescent tissue stem cells, including expression of p53 signaling genes, and are enriched for transcripts common to damage-induced quiescent revival stem cells of the regenerating intestine. In addition, we identify negative regulators of cell cycle, downstream of p53, that we show are indicators of poor prognosis and may be targeted for qCSC abolition in both p53 wild-type and mutant tumors. These data support the temporal inhibition of downstream targets of p53 signaling, in combination with standard-of-care treatments, for the elimination of qCSCs and prevention of relapse in colon cancer.

Additive Benefits of Radium-223 Dichloride and Bortezomib Combination in a Systemic Multiple Myeloma Mouse Model.

Osteolytic bone disease is a hallmark of multiple myeloma (MM) mediated by MM cell proliferation, increased osteoclast activity, and suppressed osteoblast function. The proteasome inhibitor bortezomib targets MM cells and improves bone health in MM patients. Radium-223 dichloride (radium-223), the first targeted alpha therapy approved, specifically targets bone metastases, where it disrupts the activity of both tumor cells and tumor-supporting bone cells in mouse models of breast and prostate cancer bone metastasis. We hypothesized that radium-223 and bortezomib combination treatment would have additive effects on MM. In vitro experiments revealed that the combination treatment inhibited MM cell proliferation and demonstrated additive efficacy. In the systemic, syngeneic 5TGM1 mouse MM model, both bortezomib and radium-223 decreased the osteolytic lesion area, and their combination was more effective than either monotherapy alone. Bortezomib decreased the number of osteoclasts at the tumor-bone interface, and the combination therapy resulted in almost complete eradication of osteoclasts. Furthermore, the combination therapy improved the incorporation of radium-223 into MM-bearing bone. Importantly, the combination therapy decreased tumor burden and restored body weights in MM mice. These results suggest that the combination of radium-223 with bortezomib could constitute a novel, effective therapy for MM and, in particular, myeloma bone disease.

Darolutamide Potentiates the Antitumor Efficacy of a PSMA-targeted Thorium-227 Conjugate by a Dual Mode of Action in Prostate Cancer Models.

PURPOSE: Androgen receptor (AR) inhibitors are well established in the treatment of castration-resistant prostate cancer and have recently shown efficacy also in castration-sensitive prostate cancer. Although most patients respond well to initial therapy, resistance eventually develops, and thus, more effective therapeutic approaches are needed. Prostate-specific membrane antigen (PSMA) is highly expressed in prostate cancer and presents an attractive target for radionuclide therapy. Here, we evaluated the efficacy and explored the mode of action of the PSMA-targeted thorium-227 conjugate (PSMA-TTC) BAY 2315497, an antibody-based targeted alpha-therapy, in combination with the AR inhibitor darolutamide. EXPERIMENTAL DESIGN: The in vitro and in vivo antitumor efficacy and mode of action of the combination treatment were investigated in preclinical cell line-derived and patient-derived prostate cancer xenograft models with different levels of PSMA expression. RESULTS: Darolutamide induced the expression of PSMA in androgen-sensitive VCaP and LNCaP cells in vitro, and the efficacy of darolutamide in combination with PSMA-TTC was synergistic in these cells. In vivo, the combination treatment showed synergistic antitumor efficacy in the low PSMA-expressing VCaP and in the high PSMA-expressing ST1273 prostate cancer models, and enhanced efficacy in the enzalutamide-resistant KUCaP-1 model. The treatments were well tolerated. Mode-of-action studies revealed that darolutamide induced PSMA expression, resulting in higher tumor uptake of PSMA-TTC, and consequently, higher antitumor efficacy, and impaired PSMA-TTC-mediated induction of DNA damage repair genes, potentially contributing to increased DNA damage. CONCLUSIONS: These results provide a strong rationale to investigate PSMA-TTC in combination with AR inhibitors in patients with prostate cancer.