Dominik Mumberg

Radium-223 Inhibits Osseous Prostate Cancer Growth by Dual Targeting of Cancer Cells and Bone Microenvironment in Mouse Models

Purpose: Radium-223 dichloride (radium-223, Xofigo), a targeted alpha therapy, is currently used for the treatment of patients with castration-resistant prostate cancer (CRPC) with bone metastases. This study examines the mode-of-action and antitumor efficacy of radium-223 in two prostate cancer xenograft models. Experimental Design: Mice bearing intratibial LNCaP or LuCaP 58 tumors were randomized into groups (n = 12–17) based on lesion grade and/or serum PSA level and administered radium-223 (300 kBq/kg) or vehicle, twice at 4-week intervals. X-rays and serum samples were obtained biweekly. Soft tissue tumors were observed macroscopically at sacrifice. Tibiae were analyzed by gamma counter, micro-CT, autoradiography and histology. Results: Radium-223 inhibited tumor-induced osteoblastic bone growth and protected normal bone architecture, leading to reduced bone volume in LNCaP and abiraterone-resistant LuCaP 58 models. Furthermore, radium-223 resulted in lower PSA values and reduced total tissue and tumor areas, indicating that treatment constrains prostate cancer growth in bone. In addition, radium-223 suppressed abnormal bone metabolic activity as evidenced by decreased number of osteoblasts and osteoclasts and reduced level of the bone formation marker PINP. Mode-of-action studies revealed that radium-223 was deposited in the intratumoral bone matrix. DNA double-strand breaks were induced in cancer cells within 24 hours after radium-223 treatment, and PSA levels were significantly lower 72 hours after treatment, providing further evidence of the antitumor effects. Conclusions: Taken together, radium-223 therapy exhibits a dual targeting mode-of-action that induces tumor cell death and suppresses tumor-induced pathologic bone formation in tumor microenvironment of osseous CRPC growth in mice. Clin Cancer Res; 23(15); 4335–46. ©2017 AACR.

BAY 1125976, a selective allosteric AKT1/2 inhibitor exhibits high efficacy on AKT signaling‐dependent tumor growth in mouse models

The PI3K‐AKT‐mTOR signaling cascade is activated in the majority of human cancers, and its activation also plays a key role in resistance to chemo and targeted therapeutics. In particular, in both breast and prostate cancer, increased AKT pathway activity is associated with cancer progression, treatment resistance and poor disease outcome. Here, we evaluated the activity of a novel allosteric AKT1/2 inhibitor, BAY 1125976, in biochemical, cellular mechanistic, functional and in vivo efficacy studies in a variety of tumor models. In in vitro kinase activity assays, BAY 1125976 potently and selectively inhibited the activity of full‐length AKT1 and AKT2 by binding into an allosteric binding pocket formed by kinase and PH domain. In accordance with this proposed allosteric binding mode, BAY 1125976 bound to inactive AKT1 and inhibited T308 phosphorylation by PDK1, while the activity of truncated AKT proteins lacking the pleckstrin homology domain was not inhibited. In vitro, BAY 1125976 inhibited cell proliferation in a broad panel of human cancer cell lines. Particularly high activity was observed in breast and prostate cancer cell lines expressing estrogen or androgen receptors. Furthermore, BAY 1125976 exhibited strong in vivo efficacy in both cell line and patient‐derived xenograft models such as the KPL4 breast cancer model (PIK3CAH1074R mutant), the MCF7 and HBCx‐2 breast cancer models and the AKTE17K mutant driven prostate cancer (LAPC‐4) and anal cancer (AXF 984) models. These findings indicate that BAY 1125976 is a potent and highly selective allosteric AKT1/2 inhibitor that targets tumors displaying PI3K/AKT/mTOR pathway activation, providing opportunities for the clinical development of new, effective treatments.

Identification of Atuveciclib (BAY 1143572), the First Highly Selective, Clinical PTEFb/CDK9 Inhibitor for the Treatment of Cancer

Selective inhibition of exclusively transcription‐regulating PTEFb/CDK9 is a promising new approach in cancer therapy. Starting from lead compound BAY‐958, lead optimization efforts strictly focusing on kinase selectivity, physicochemical and DMPK properties finally led to the identification of the orally available clinical candidate atuveciclib (BAY 1143572). Structurally characterized by an unusual benzyl sulfoximine group, BAY 1143572 exhibited the best overall profile in vitro and in vivo, including high efficacy and good tolerability in xenograft models in mice and rats. BAY 1143572 is the first potent and highly selective PTEFb/CDK9 inhibitor to enter clinical trials for the treatment of cancer.

MOLECULAR MECHANISMS AND COMBINATION STRATEGIES WITH PI3K AND BTK INHIBITORS TO OVERCOME INTRINSIC AND ACQUIRED RESISTANCE IN PRECLINICAL MODELS OF ABC-DLBCL

The Brutons tyrosine kinase (Btk) inhibitor ibrutinib has demonstrated promising efficacy in a variety of hematologic malignancies. However, the precise mechanism of action of the drug remains to be fully elucidated. Tumor‐infiltrating macrophages presented in the tumor microenvironment have been shown to promote development and progression of B‐cell lymphomas through cross talk mediated by secreted cytokines and chemokines. Because Btk has been implicated inToll‐like receptor (TLR) signaling pathways that regulate macrophage activation and production of proinflammatory cytokines, we investigate the immunomodulatory effects of Btk inhibitor on macrophages. Our results demonstrate that Btk inhibition efficiently suppresses production of CXCL12, CXCL13, CCL19, and VEGF by macrophages. Furthermore, attenuated secretion of homeostatic chemokines from Btk inhibitor‐treated macrophages significantly compromise adhesion, invasion, and migration of lymphoid malignant cells and even those not driven by Btk expression. The supernatants from Btk inhibitor‐treated macrophages also impair the ability of endothelial cells to undergo angiogenic tube formation. Mechanistic analysis revealed that Btk inhibitors treatment downregulates secretion of homeostatic chemokines and cytokines through inactivation of Btk signaling and the downstream transcription factors, NF‐κB, STAT3, and AP‐1. Taken together, these results suggest that the encouraging therapeutic efficacy of Btk inhibitor may be due to both direct cytotoxic effects on malignant B cells and immunomodulatory effects on macrophages present in the tumor microenvironment. This novel mechanism of action suggests that, in addition to B‐cell lymphomas, Btk inhibitor may also have therapeutic value in lymphatic malignancies and solid tumors lacking Btk expression.

Abstract 321: Synergistic activity of the ATR inhibitor BAY 1895344 in combination with DNA damage inducing and DNA repair compromising therapies in preclinical tumor models

The DNA damage response (DDR) system consists of complex signalling pathways that secure the integrity of the genome in eukaryotic cells. DDR pathway activation follows recognition of DNA damage and results in cell cycle arrest, suppression of general translation, induction of DNA repair, cell survival or even cell death. Proteins that directly recognize aberrant DNA structures recruit and activate kinases of the DDR, such as ATR (ataxia telangiectasia and Rad3-related). ATR responds to a broad spectrum of DNA damages, including double-strand breaks (DSB) and lesions derived from interference with DNA replication as well as increased replication stress. Therefore, inhibition of ATR kinase activity could be the basis for a novel anti-cancer therapy in tumors with increased DNA damage, deficiency in DDR or replication stress. The potential of combining ATR kinase inhibitor with DNA damage inducing or DNA repair compromising anti-cancer therapeutics was studied in preclinical tumor models. We assessed the novel ATR kinase inhibitor (ATRi) BAY 1895344 in combination with external beam radiation therapy (EBRT), poly ADP ribose polymerase (PARP) inhibition or anti-androgen (AA) therapy. In cellular anti-proliferation assays as well as in tumor xenograft studies we could demonstrate synergistic activity of BAY 1895344 in combination treatment with the PARP inhibitor AZD-2281 in the homologous recombination (HR) defective breast cancer model MDA-MB-436, and with the non-steroidal AA darolutamide in the hormone-dependent prostate cancer model LAPC-4. Strong synergistic anti-tumor activity of BAY 1895344 could be further demonstrated in combination with EBRT inducing long-lasting tumor growth inhibition in the colorectal cancer xenograft model LOVO. The mechanism-based potential of combining DNA damage induction by EBRT with ATRi BAY 1895344 suggests a potential new treatment option for radiation therapy-resistant patients. Furthermore, the inhibition of parallel DDR pathways, as a combination of ATRi BAY 1895344 with a PARP inhibitor, indicates novel treatment opportunities in breast cancer patients with homologous recombination deficiencies, as does the synergism of BAY 1895344 and AA darolutamide therapy in hormone-dependent prostate cancer patients. BAY 1895344 is currently under clinical investigation in patients with advanced solid tumors and lymphomas (NCT03188965). Citation Format: Antje Margret Wengner, Gerhard Siemeister, Ulrich Luecking, Julien Lefranc, Kirstin Meyer, Eleni Lagkadinou, Bernard Haendler, Pascale Lejeune, Dominik Mumberg. Synergistic activity of the ATR inhibitor BAY 1895344 in combination with DNA damage inducing and DNA repair compromising therapies in preclinical tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 321.

Abstract 855: Increased in vitro potency and in vivo efficacy of FGFR2-targeted thorium-227 conjugate (FGFR2-TTC) in combination with the ATR inhibitor BAY 1895344

Targeted Thorium-227 Conjugates (TTCs) consist of the alpha emitter Thorium-227 (227Th) coupled, by a 3, 2-HOPO chelator, to a tumor specific antibody. The alpha particles release high energy over a short range (2- 10 cell diameters), resulting in a potent local irradiation of the tumor with limited damage to surrounding tissue. Here, we describe the in vitro and in vivo evaluation of an FGFR2 targeted thorium-227 conjugate (FGFR2-TTC) in combination with the ATR inhibitor BAY 1895344. FGFR2 (fibroblast growth factor receptor 2) is a receptor tyrosine kinase and overexpression of FGFR2 has been described in different cancers, while its expression in healthy human tissues is moderate to low. This renders FGFR2 an attractive antigen to explore the concept of targeted alpha therapy (TAT). The mode-of-action of TTCs is based on the induction of clustered DNA double strand breaks and G2 cell cycle arrest. We hypothesized that combination of FGFR2-TTC with inhibitors of DNA damage response (DDRi9s) may enhance potency and efficacy. The ataxia telangiectasia and Rad3-related protein (ATR) kinase is a central mediator of DDR. ATR kinase responds to a broad spectrum of DNA damage, including double-strand breaks (DSB) and lesions derived from interference with DNA replication as well as increased replication stress. Inhibition of ATR kinase activity induces cell death especially in tumors with increased DNA damage, deficiency in DNA damage repair or replication stress. Therefore, we investigated whether the combination of the FGFR2-TTC with the ATRi BAY 1895344 results in enhanced tumor sensitivity in vitro and in vivo. In in vitro cytotoxicity assays, the combination of FGFR2-TTC and BAY 1895344 resulted in increased potency of the FGFR2-TTC on three different cancer cell lines (KATO III (gastric), MFM-223 (triple negative breast cancer), SUM52-PE (triple negative breast cancer)). Mechanistic analysis demonstrated that the combination treatment resulted in reduced levels of G2 arrest and increased levels of DNA damage in comparison to single agent treatments. The combination was further evaluated in vivo using the MFM-223 breast cancer xenograft model. An increased anti-tumor efficacy of a low dose of FGFR2-TTC (100 kBq/kg) was observed in combination with BAY 1895344 compared to animals treated with vehicle. The presented data support the mechanism-based rationale for combining DNA damage induction by FGFR2-TTC with DNA damage repair inhibition using ATRi BAY 1895344. Our findings warrant further exploration of TTCs in combination with BAY 1895344 for cancer therapy. Citation Format: Katrine Wickstroem, Urs B. Hagemann, Antje M. Wengner, Anette Sommer, Alexander Kristian, Christine Ellingsen, Roger M. Bjerke, Jenny Karlsson, Olav B. Ryan, Lars Linden, Bertolt Kreft, Dominik Mumberg, Hanno Wild, Karl Ziegelbauer, Alan Cuthbertson. Increased in vitro potency and in vivo efficacy of FGFR2-targeted thorium-227 conjugate (FGFR2-TTC) in combination with the ATR inhibitor BAY 1895344 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 855.

COPANLISIB IN COMBINATION WITH ANTI‐PD‐1 INDUCES REGRESSION IN ANIMAL TUMOR MODELS INSENSITIVE OR RESISTANT TO THE MONOTHERAPIES OF PI3K AND CHECKPOINT INHIBITORS

KARPAS299, KI‐JK), PTCL (FEPD, HH) and SS (H9, HUT78) were exposed to increasing doses of copanlisib alone and in combination with increasing doses of other compounds using the fixed ratio set‐ up. Tested compounds were anti CD30 antibody‐drug conjugate brentuximab, ALK‐i crizotinib, CDK‐i roniciclib, DNA damage agent bendamustine, HDAC‐i panobinostat and romidepsin, immunomodulatory lenalidomide, JAK1/2‐i ruxolitinib, BTK‐i ibrutinib, MALT‐i MI2, proteasome‐i bortezomib, BCL2‐i venetoclax, CDK4/6‐i palbociclib, the BET‐i BAY 1238097, and the PTEFb/CDK9‐i BAY 1143572. Synergy was assessed with Chou‐Talalay combination index (CI): synergism (<0.9), additive (0.9–1.1), antagonism/no benefit (>1.1). Gene expression profiling was done using the Illumina‐HumanHT‐12 Expression‐BeadChips and GSEA (FDR < 0.25). Results: Copanlisib had a median IC50 of 285 nM (50–1660 nM). Among the other compounds, the most active were bortezomib (IC50 3.1 nM; 1.6–6 nM), romidepsin (IC50 2.4 nM; 1.8–7.7 nM), panobinostat (IC50 10.2 nM, 3.8–14 nM), roniciclib (IC50 21.1 nM, 13.4–50.1 nM). Different copanlisib‐containing combinations, tested in the 9 cell lines, were synergistic: copanlisib with palbociclib (7/9 cell lines), panobinostat (7/9), BAY 1238097 (6/9), venetoclax (5/9), romidepsin (5/9), ruxolitinib (4/9), lenalidomide or BAY 1143572 or brentuximab or crizotinib (3/9). The most promising combinations were copanlisib/venetoclax and copanlisib/palbociclib, with a median CI < 0.7 in 3 cell lines. High expression of genes involved in interferon signaling and TP53 pathway were associated with synergism to copanlisib/venetoclax, while MYC target genes and cell cycle signaling were associated with resistance to the combination. Largely, the opposite was observed for copanlisib/palbociclib, with synergism in cells with high expression of E2F targets and genes involved in cell cycle and resistant in cells with expression of transcripts involved in interferon and TP53 signaling. Conclusion: Copanlisib was active in T‐cell lines derived from ALCL, PTCL and SS. The combinations with the BCL2‐inhibitor venetoclax and with the CDK4/CDK6‐inhibitor were the most synergistic and the specific GEP features might predict lymphomas that could benefit from these regimens.

Abstract 1079: Preclinical activity of the FGFRinhibitor BAY 1163877 alone or in combination with antihormonal therapy in breast cancer

BAY 1163877 is an orally available, highly potent and selective pan fibroblast growth factor receptor (FGFR) inhibitor. In an ongoing Phase 1 clinical trial (NCT01976741) BAY 1163877 showed clinical responses at exceptional tolerability in patients suffering from different tumor types including urothelial bladder carcinoma or lung tumors, which were selected based on elevated FGFR1-3 mRNA expression. In the preclinical phase, the compound demonstrated significant single agent anti-tumor activity in various tumor models with different FGFR alterations leading to FGFR overexpression (e.g. FGFR gene amplifications or mutations). Genetic alterations of FGFRs can also be found in breast cancer with 7.5 - 17% of all tumors harboring a FGFR1 gene amplification. Elevated FGFR1 mRNA levels can be found in up to 22% of breast cancer cell lines as well as clinical samples. Other FGFR alterations include FGFR2 or FGFR4 gene amplifications as well as elevated FGFR mRNA levels, which were reported in all breast cancer subtypes. We therefore investigated BAY1163877 monotherapy in various breast cancer models. Due to the favorable clinical safety profile of BAY1163877, we also examined a combination treatment with early line antihormonal therapies in hormone receptor positive breast cancer. In vitro profiling of BAY 1163877 in a number of breast cancer cell lines showed a clear association of efficacy with expression levels of different FGFR isoforms. The efficacy was further investigated in several patient- or cell line-derived breast cancer in vivo models. For instance, BAY 1163877 alone dosed 38mg/kg twice daily induced tumor growth inhibition of greater than 90% in a subcutaneous mouse syngeneic 4T1 breast cancer model expressing elevated levels of FGFR2. Resistance to endocrine therapy appears associated with FGFR1 gene amplification and may explain the poor prognosis of FGFR1 overexpressing tumors treated with adjuvant tamoxifen. We therefore investigated the combination of the panFGFR-inhibitor BAY 1163877 with the clinically used antihormonal compound fulvestrant in selected luminal breast cancer PDX models. Some of these models showed resistance to antihormonal treatment in monotherapy but improved in vivo efficacy in combined treatment using BAY 1163877 and fulvestrant. These data may warrant further clinical investigation of BAY1163877alone or in combination with antihormonal therapy in patients with FGFR overexpressing breast cancer. Citation Format: Oliver Politz, Peter Ellinghaus, Sebastian Bender, Sylvia Gruenewald, Franziska Siegel, Marie-Pierre Collin, Sabine Zitzmann-Kolbe, Dominik Mumberg, Karl Ziegelbauer. Preclinical activity of the FGFRinhibitor BAY 1163877 alone or in combination with antihormonal therapy in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1079. doi:10.1158/1538-7445.AM2017-1079