FIGURE IV A) By using the Incucyte® Cell Cycle Lentivirus, a single-cassette, genetically encoded ubiquitination-based indicator that takes advantage of cell cycle dependent changes in the expression patterns of Geminin and Cdt1 stable cell lines were produced. Fluorescent proteins TagGFP2 and mKate2 (green/red), or TagGFP2 and TagRFP (green/orange) were linked to fragments of Geminin and Cdt1. G1 (red), the transition from G1 to S (yellow) and S/G2/M (green) phases were monitored in realtime for an untreated (B) or an 1000 nM OMX- 0407 treated RCC cell line (C). D) Representative images of untreated and 4h OMX-0407 (1000 nM) treated renal cancer cells. E) OMX-0407 dose-dependent growth kinetics analyzed by phase confluence in a live cell imaging system. OMX-0407 dose-dependent growth (F) and G1 / G1-S arrest (G) of representative cell lines from different indications. G) OMX-0407 dose-dependent inhibition of cells from different indications in G1 / G1-S arrest upon OMX-0407 treatment. Phospho-protein Western Blot analyses of Caki-1 renal cancer cell lysates. Representative images of blots for SFK-Y530 (upper band), pPAK1/2/3 (lower band) (H), CDC42 (upper band) and pERK1/2 (lower band) (I) pre-and post treatment with OMX-0407. J) Signal to noise ratio after total protein normaization of pSFK-Y530, pPAK1/2/3, CDC42 and pERK1/2. K) Lumit® Immunoassay detection kit for pERK1 (Thr202) in Caki-1 renal cancer cells with different concentrations of OMX-0407 or DMSO as control (ctrl.), data shown as fold change normalized to control. → OMX-0407, a potent spectrum-selective kinase inhibitor demonstrates outstanding anti-tumor efficacy in vitro as well as ex vivo in a variety of cancer cell lines across different indications. → OMX-0407 shows remarkable efficacy both as a monotherapy and in combination with VEGFR2 inhibition in the RENCA tumor model, supporting the potential of OMX-0407 in the treatment of RCC patients. → OMX-0407 treatment arrests tumor cells in vitro and in vivo by inhibiting both cell cycle and proliferation. → The ongoing comprehensive studies on the mechanism of action of OMX-0407 in inhibiting tumor cell proliferation will guide the further clinical evaluation of OMX-0407, which is currently being investigated in the ongoing first-in-human trial OMX-0407-101 (NCT05826600). Conclusion OMX-0407, a spectrum-selective kinase inhibitor drives cell-cycle arrest in vitro and in vivo – an in-depth MoA analysis by phospho-proteomics Ilona-Petra Maser 1 , Marisa Stebegg-Wagner 1, Bettina Bauer 1, Andreas Schirmer 1, Moritz Zulley 1, Filippos Konstantinidis 1, Sonja Lacher 1, Barbara Kracher 2, Parastou Kohvaei 2, Marti Bernardo 3, Oleg Deryagin 3, Simar Pal Singh 4, Murray Yule 1, Hannes Loferer 1 and Stefan Bissinger 1 1 iOmx Therapeutics AG, Martinsried/Munich, Germany; 2 Evotec GmbH, Neuried/Munich, Germany; 3 Clarivate PLC, London, UK; 4 PamGene International B.V., 's-Hertogenbosch, The Netherlands Authors and affiliations Introduction FIGURE II A) Overlap of differentially regulated phospho-sites between CRC/Gastric and adeno NSCLC cancer cell lines. B) Overlap of predicted upstream kinases between CRC/Gastric and adeno NSCLC cancer cell lines. C) Kinase volcano Plot of OMX-0407 sensitive adeno NSCLC cancer cell lines showing the effect size and direction (x-axis, Median kinase statistic) versus significance and specificity (y-axis, Median final score) upon OMX-0407 treatment. Median Kinase Statistic is the median change in set of peptides linked to a kinase and represents change in kinase activity in a treatment (T) compared to a untreated control (U), where < 0 means inhibition, > 0 means activation in T versus U. Median final score is the sum of the Kinase Significance Score and the Kinase Specificity Score, where Kinase Significance Score is the permutation test that predicts probability of a kinase being differentially active between T and U, while Kinase Specificity Score is the permutation test that predicts probability of a set of peptides linked to a kinase. Blue spots in volcano plot are kinases that show significant difference (i.e. Median final score -log10(p)>1.3 or p<0.05) compared to untreated control. D)Heatmap comparing phosphosites in adeno- NSCLC cell lines before and after OMX-0407 treatment. Tumor cell viability screening showed distinct sensitivity profile upon OMX-0407 therapy → Anti-tumor monotherapy efficacy screening of OMX-0407 in 225 human cancer cell lines identifies a sensitivity profile of individual cell lines towards OMX-0407 → Squamous non-small cell lung cancer and renal cell carcinoma are emerging as key tumor indications responding to OMX-0407 FIGURE III Schematic representation of the different signaling pathways involved in OMX-0407 mediated cell cycle arrest. The figure shows the different key pathways, such as RAS/RAF, MAPK, PI3K/AKT/mTOR, involved in the manifestation of the OMX-0407 mediated cell cycle arrest. OMX-0407 inhibits tumor cell growth by blocking the G1 to S transition, resulting in cell cycle arrest → OMX-0407 induces a significant dose-dependent inhibition of tumor cell growth in vitro across many indications → OMX-0407 significantly arrests cancer cells in G1 / G1-S phase as demonstrated by live cell imaging of cell cycle dynamics over time → OMX-0407 dependent cell cycle arrest is associated with the inactivation or downregulation of key regulators of the cell cycle, such as SFKs, pPAKs, pERK1/2 or CDC42 FIGURE V RENCA tumor cells were subcutaneously implanted and randomized at an average tumor volume of 130 mm³. Tumor bearing animals were treated twice daily with both 50 or 25 mg/kg OMX-0407 and the appropriate vehicle control for Axitinib and 12.5 mg/kg Axitinib or the appropriate vehicle control for OMX-0407 in combination by oral gavage. A) Tumor growth kinetics of the efficacy animals. Average tumor growth is depicted as mean ± SEM for n≤12 mice per therapy group by using the last observation carried forward method. B) Tumor growth kinetics of separate randomized cohorts terminated for tissue sampling after 7 days of therapy. Sampling of EDTA-plasma, peripheral blood mononuclear cells (PBMCs) and tumor tissue for pharmacodynamics analyses shown in Figure IV C-H. C) OMX-0407 plasma concentration. D) Flow cytometry analysis of median fluorescence intensity of phospho-SFK-Y530 in monocytes of isolated PBMCs. E) Representative Western blot protein detection for phospho SFK-Y530 (upper row) and phospho PAK1/2/3-S144/S141/S139 (lower row). F) Signal to noise ratio after total protein normalization of anti-human p-PAK1/2/3; SFK-Y530 and pERK1/2 G) Quantification of percentage Ki-67⁺ area in viable tumor cell area. Individual data points represent the average of 5 independent field of views of a KI-67 immunoflurescence staining. I) Representative images of immunofluorescence staining of proliferation marker Ki-67 in tumor sections. Data of C, E, F , G, H are shown as mean ± SEM for n= 6 animals per treatment group. FIGURE I A) Schematic overview of a high thoughput tumor cell viability screening. Each cell line was treated for 48 h with OMX-0407 (6 µM – 4 nM), cisplatin a standard chemotherapy drug (1 mM – 100 nM) as reference control and OMX-0407-vehicle control in a 9-point serial dilution. Tumor cell viability was analyzed using the CellTiter-Glo® assay according to the manufacturer's protocol. Dose-response curves were fitted using nonlinear, sigmoidal regression models. Absolute IC50 values were calculated according to dose-response curves generated by GraphPad Prism 5.0. B) 225 human cancer cell lines of 15 different indications were analyzed and sensitivity scores were calculated by the following formula: Sensitivity score = Log 2(2 x %max Inhibition)² – Log 2(IC50abs) + 2.5; sensitivity score > 0 = sensitive to OMX-0407; sensitivity score < 0 = resistant to OMX-0407. RCC = renal cell carcinoma, sqNSCLC = squamous non-small cell lung cancer . OMX-0407 inhibits tyrosine kinases involved in cytoskeletal rearrangement, cell proliferation and cell cycle → OMX-0407 targets receptor tyrosine kinase families that are highly relevant for tumor cell proliferation → OMX-0407 targets multiple levels within a signaling pathway ensuring a comprehensive inhibition of cancer cell growth by blocking cell proliferation and inducing a cell cycle arrest → By targeting more than one relevant pathway of cancer cell proliferation and survival, OMX-0407 mitigates the risk of redundant pathways and the out-growth of cancer cells Phospho-proteomics analysis reveals significant dephosphorylation of key cancer signaling pathways upon OMX-0407 treatment → Phospho-proteomics and kinase activity measurements reveal significant OMX-0407 activity in cancer cell lines → Colorectal cancer (CRC)/gastric and adeno NSCLC cancer cell lines were treated for 4h with 200nM OMX-0407 and subjected to phospho-proteomics analysis by mass spectrometry and kinase activity measurements using multiplex, functional kinase activity profiling → Both analyses demonstrate an OMX-0407-mediated reduction in the phosphorylation levels of many phosphosites. PamGene analysis reveals that 68% (90/133) of OMX-0407 dephosphorylated phospho-sites overlap between CRC and NSCLC cell lines → Based on upstream kinase activity predictions, most of the 63% (73/115) OMX-0407-deactivated kinases in CRC and NSCLC cell lines are part of the the pro-tumorigenic SFK. OMX-0407, an orally available, spectrum-selective kinase inhibitor targets key members of the Salt-inducible kinase (SIK) and tyrosine (-like) kinase families. An anti-tumor viability screen of >200 human cancer cell lines revealed striking effects of OMX-0407 across various cancer indications, with particularly strong effects on renal cell carcinoma (RCC) and squamous non-small cell lung cancer (sqNSCLC). Phospho-proteomics and multiplex, functional kinase activity profiling demonstrate significant anti-tumor activity of OMX-0407 via simultaneous inhibition of key cellular processes such as cell proliferation and cell cycle regulation. Orthogonal functional in vitro assays confirmed the association of the OMX-0407-mediated anti-tumor efficacy with the arrest of G1/ S transition and corresponding dephosphorylation of cell cycle-associated proteins downstream of the Src family kinases (SFK) and the serine/theronine kinases PAK1/2. The inhibitory effects of OMX-0407 on cell proliferation and cell cycle regulation result in potent anti-tumor efficacy of OMX-0407 as a single agent in the RENCA-RCC mouse model, which is further enhanced in combination with axitinib, an inhibitor of the vascular endothelial growth factor receptor 2 (VEGFR2). These data further strengthen the potential of OMX-0407 for the treatment of RCC as well as other indications. In summary, OMX- 0407 is a novel spectrum-selective kinase inhibitor for patients with high unmet medical need, that is currently being evaluated in a clinical Phase I trial (NCT05826600). AACR 2024 #514 Results Remarkable anti-tumor efficacy of OMX-0407 in the syngeneic RCC tumor model → OMX-0407 treatment significantly inhibits tumor growth in the RENCA RCC mouse model. This effect is further enhanced in combination with the anti-angiogenic therapy Axitinib targeting vascular endothelial growth factor receptor 2 (VEGFR2) → Tumor growth inhibition by OMX-0407 was associated with dose-dependent reduction in the phosphorylation of SFKs & pPAKs both in the tumor and peripheral blood mononuclear cells, which correlated well with OMX-0407 plasma levels. → OMX-0407 treatment resulted in a significant reduction of Ki67+ proliferating cells within the tumor area, likely driven by OMX- 0407-induced cancer cell cycle arrest. A) High throughput screening of cancer cell viability with OMX-0407 B) Distinct activity profile of OMX-0407 on tumor cell lines Indication4 RCC sqNSCLC Indication 20 Indication 25 Indication2 Indication1 Indication 26 Indication 13 Indication 16 Indication 10 Indication 22 Indication 32 Indication 29 Indication 37 -10 -5 0 5 10 Sensi�vity Scores for OMX-0407 ac�vity Sensi�ve Resistant In vitro cell line treatment Luminescence imaging L Cell line 1 -3 -2 -1 0 1 2 3 -20 0 20 40 60 80 100 120 OMX-0407 Cisplatin log drug [µM] % of surviving cells absolut IC50 An�-tumor ac�vity KDRRon Lyn Src Etk/BMXAbl 1 2 3 4 5 −2 −1 0 1 2 Median Kinase sta�s�cs (Effect size & Direc�onality) Median Final Score (Significance & Specificity) Change Unchanged Decreased Cell line 1 Cell line 2 Cell line 3 Cell line 4 Cell line 5 Cell line 6 Cell line 7 Cell line 8 Cell line 9 Cell line 1 Cell line 2 Cell line 3 Cell line 4 Cell line 5 Cell line 6 Cell line 7 Cell line 8 Cell line 9 Untreated 4h OMX-0407 (200nM) A B C D Contact Ras Raf MEK1/2 ERK1/2 RRTTKK SOSSHC GRB2 PKCPAK1/2 Paxillin PIP2 SRC CCyyttoosskkeelleettoonn rreeaarrrraannggeemmeenntt aanndd cceellll mmiiggrraatitioonn CCeellll pprroolliiffeerraatitioonn CCeellll ssuurrvviivvaall Cdk4/6 Cdk2 Cyc D c-Myc mTORC1 AKT TSC1/2 FYN PP STAT3 STAT3 P STAT3 P RRTTKK RB1MM SSGG22 GG11 GG00 pphhaassee (resting) G1/S checkpoint M/G1 checkpoint CDK4/6 Cyclin D p16 CDK2 Cyclin E p21 OOMMXX--00440077OOMMXX--00440077 CCeellll ccyyccllee ARHGAP35 CDC42 Vincullin If not indicated otherwise, graphs are representative data from at least two independent experiments. Sigmoidal dose–response curves were fitted to data using 4-Parameter Logistic (4PL) non-linear curve models for in vitro dose responses. Data points show mean ± SEM. Significance was calculated by two-way ANOVA analysis including Tukeys multiple comparison analysis. A B C D E G Vehicle Axi�nib OMX-0407 (50mg/kg)OMX-0407 (25mg/kg) OMX-0407 (50mg/kg) + Axi�nib Vehicle 1 Axitinib OMX-0407 (25mg/kg) OMX-0407 (50mg/kg) OMX-0407 (50 mg/kg) & Axitinib F H 0 2×10 4 4×10 4 6×10 4 pSFK-Y530 Median Fluorescence intensity 0 500 1000 1500 2000 2500 Plasma level OMX-0407 concentra�on (nM) 25 mg/kg 50 mg/kg 50 mg/kg Vehicle 50 mg/kg + Axi�nib Axi�nib Vehicle Axi�nib 50 mg/kg + Axi�nib pSFK_Y530 ~59kDa pPAK1/3 ~68kDa pPAK2 ~61ka OMX-0407 pERK1/2 ~42 / 44 kDa RCC Acute study 02468 1 0 0 200 400 600 800 Days after start of treatment Tumor volume (mm³) mean ± SEM RCC Efficacy study 0 5 10 15 20 25 30 35 0 500 1000 1500 Days after start of treatment Tumor volume (mm³) mean ± SEM -100 -75 -50 -25 0 25 50 pPAK1/2/3 Change rel. to ctrl. [%] 0.0220 -100 -75 -50 -25 0 25 50 pSFK-Y530 Change rel. to ctrl. [%] 0.017 <0.0001 <0.0001 <0.0001 -100 -75 -50 -25 0 25 50 pERK1/2 Change rel. to ctrl. [%] 0% 25% 50% 75% 100% 125% Tumor cell prolifera�on % KI67+ area / tumor area 0.01 0.001 <0.001 Vehicle ctrl. OMX-0407 (50 mg/kg) Axi�nib OMX-0407 50 mg/kg + Axi�nib OMX-0407 (25 mg/kg) 0.01 0.001 <0.001 UntreatedOMX-0407 (1000nM) 0h 32h DMSO 25nM 50nM 100nM OMX-0407 pSFK_Y530 ~59 kDa pPAK1/3 ~68 kDa DMSO 1000nM pERK1/2 ~44/42 kDa CDC42 ~21 kDa DMSO 25nM 100nM 1000nM 0.0 0.2 0.4 0.6 pSFK_Y530 OMX-0407 signal - protein normalized DMSO 25nM 100nM 1000nM 0.0 0.2 0.4 0.6 0.8 pPAK1/2/3 OMX-0407 signal - protein normalized DMSOCtrl OMX-0407 0.00 0.02 0.04 0.06 0.08 0.10 pERK1/2 signal - protein normalized DMSOCtrl OMX-0407 0.00 0.05 0.10 0.15 0.20 0.25 CDC42 signal - protein normalized 0 20 40 60 80 100 101 102 103 Growth arrest (48h a�er drug administra�on) conc. OMX-0407 nM Phase confluence [%] DM SO Lova 0 20 40 60 80 100 101 102 103 G1 + G1-S arrest (48h a�er drug administra�on) conc. OMX-0407 nM Cells in cell cycle arrest [%] DM SO Lova NSCLC RCC MESO MEL 0 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48 0 20 40 60 Cell Cycle (Vehicle) Time [h] Population [%] 0 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48 0 20 40 60 80 Cell Cycle (1000 nM OMX-0407) Time [h] Population [%] S/G2/M M-G1 G1 G1-S 0 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 0 20 40 60 80 100 Cell Cycle phase confluence Phase confluence [%] 1000 nM OMX-0407 500 nM OMX-0407 250 nM OMX-0407 125 nM OMX-0407 62.5 nM OMX-0407 31.25 nM OMX-0407 DMSO ctrl. untreated (no DMSO) �me [h] A B C D E F G H I J K -100 -80 -60 -40 -20 0 pERK1 OMX-0407 [nM] Change rel. to ctrl. [%] 156,25 312,5 625 1250 2500 5000
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