OMX-0407 Synergy with PD-1

SIK3 inhibits death receptor mediated apoptosis in tumor cells  → Knockout of SIK3 sensitizes tumor cells to death receptor mediated apoptosis and thereby delays tumor cell growth Using the iOTarg genetic screening platform, salt-inducible kinase 3 (SIK3) was recently identified as a novel epigenetic modulator in cancer therapy. SIK3, a serine/threonine kinase of the AMP-activated protein kinase (AMPK) family, is known for regulating the NF-κB-driven gene landscape through phosphorylation of class IIa histone deacetylases (HDACs), causing the tumor to evade death receptor-mediated killing. Here we report that OMX-0407, an orally available, single-digit nanomolar inhibitor of SIK3 abates downstream pro-survival signaling of the SIK3-HDAC4-NF-κB axis and potentiates caspase-mediated cell death in murine and human tumor cell lines. OMX-0407 dose-dependent suppression of intratumoral NF-κB activity was shown in vitro and in vivo with an MC38 NF-κB-luc reporter cell line. OMX-0407 monotherapy results to significant tumor growth inhibition (TGI) as well as prolonged survival in the highly immune-infiltrated syngeneic murine colorectal carcinoma model MC38. Besides its direct inhibitory effects on cancer cells, OMX-0407 repolarizes the tumor microenvironment (TME) by strongly decreasing regulatory T cells (Treg) and M2-polarized macrophages in the tumor bed, while not affecting the peripheral T cell compartment. Using immune checkpoint inhibitor resistant breast (EMT6) and lung (KLN205) cancer models, we demonstrate that OMX-0407 and anti-PD-1 therapy act synergistically by combining the sensitization towards cell death with a reduction in immunosuppressive TME and an increase in cytotoxic T cell activity. OMX-0407 is an outstanding therapeutic option to overcome TNF/TRAIL-induced immune evasion and anti-PD-1 resistance, which will be clinically investigated in the near future.  → OMX-0407 is a strong inhibitor of SIK3 kinase that inhibits the phosphorylation of HDAC4 in a dose- dependent manner and thereby abrogates the nuclear activity of pro-tumorigenic transcription factor NF-κB in tumor cells both in vitro and in vivo  → Downregulation of the SIK3-HDAC4-NF-κB pathway with OMX-0407 potentiates apoptosis by death receptor ligands, such as TNF or TRAIL, in vitro in tumor cell lines of different origin  → Pharmacokinetics of OMX-0407 in tumor tissue correlates with intra-tumoral abrogation of the HDAC4- NF-κB axis  → OMX-0407 shows strong efficacy as monotherapy in the syngeneic tumor model MC38 with a marked repolarization of the tumor microenvironment towards an anti-tumor immune profile  → OMX-0407 acts synergistically with PD-1 blockade in immune checkpoint inhibitor-resistant syngeneic breast and lung cancer models  → The ability of OMX-0407 to repolarize the tumor microenvironment and sensitize tumors to death receptor-mediated apoptosis holds great potential for patients with high unmet medical need, both as monotherapy and in combination with anti-PD-1/PD-L1 immune checkpoint blockade  → OMX-0407 will enter clinical trials in patients with advanced cancer in 2022 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 with two-way ANOVA analysis including Tukeys multiple comparison analysis. Conclusion Results Statistical information Contact OMX-0407, a highly potent SIK3 inhibitor, sensitizes tumor cells to cell death and eradicates immune-checkpoint resistant tumors synergistically in combination with PD-1 inhibition Ilona-Petra Maser1, Christina Hartl1, Tillmann Michels1, Ronny Milde1, Vanessa Klein1, Michail Maraslis1 Philipp Beckhove2, Nisit Khandelwal1, Murray Yule1, Hannes Loferer1 and Stefan Bissinger1 1 iOmx Therapeutics, Martinsried/Munich, Germany 2 Regensburg Center for Interventional Immunology (RCI), Regensburg, Germany Research Authors and affiliations Introduction 0 10 20 30 40 50 0% 200% 400% 600% 800% 1000% �me [h] % Tumor cell growth [normalized to 0h] 0 ng/ml TNF SIK3wt. SIK3-KO 0 10 20 30 40 50 0% 200% 400% 600% 800% 1000% �me [h] 1 ng/ml TNF p<0.0001 SIK3wt. SIK3-KO MC-38wt. bulk MC-38wt. clone 1B1 MC-38 SIK3 KO clone 10F1 MC-38wt. bulk MC-38wt. clone 1B1 MC-38 SIK3 KO clone 10F1 A) SIK3-KO inhibits MC38 tumor cell growth in a TNF-dose dependent manner B) SIK3-KO enhances death receptor mediated tumor cell death MC38wt. cloneMC38wt. bulk MC38-SIK3-KO clone 0 1000 2000 3000 4000 Phospha�dylserine Expression (9h a�er therapy) Annexin V increase [%] 0 ng/ml TNF 1 ng/ml TNF p<0.0001 MC-38wt. bulk MC-38wt. clone 1B1 MC-38 SIK3 KO clone 10F1 0 2×10 5 4×10 5 6×10 5 8×10 5 Caspase 3/7 cleavage (8h a�er OMX-0407 treatment) Caspase 3/7 cleavage (RLU) 0 ng/ml TNF 1 ng/ml TNF p<0.0001 MC-38wt. bulk MC-38wt. clone 1B1 MC-38 SIK3 KO clone 10F1 MC38wt. cloneMC38wt. bulk MC38-SIK3-KO clone OMX-0407 inhibits SIK3-triggered phosphorylation of HDAC4/5 and the associated transcriptional activity of NF-κB  → OMX-0407 demonstrates dose dependent dephosphorylation of histone deacetylase 4/5  → OMX-0407 prevents TNF mediated actvity of the NF-κB pathway FIGURE I MC38-SIK3-KO or MC38 wildtype control clones were generated using CRISPR. MC-38-SIK3-KO and wildtype clone as well as MC38 wildtype bulk tumor cells were treated with 1 ng/ml TNF or corresponding controls. A) Tumor growth kinetics was monitored for 48h in a life cell imaging system including analysis of phosphatidylserine expression by Annexin V and caspase 3/7 cleavage 8h after therapy start (B). OMX-0407 increases TNF and TRAIL induced cleavage of caspase-3/7, cell surface phosphatidylserine expression and cell membrane disrupture of tumor cells 10 100 1000 10000 0 1×105 2×105 3×105 Caspase 3/7 cleavage (8h a�er OMX-0407 treatment) OMX-407 [nM] Caspase-3 /7 cleavage (RLU) 0 ng/ml TNF 1 ng/ml TNF DMSO-ctrl. DMSO-ctrl. 10 100 1000 10000 0 500 1000 1500 2000 Phospha�dylserine expression (14h a�er OMX-0407 treatment) OMX-407 [nM] Annexin V increase [%] 0 ng/ml TNF 1 ng/ml TNF DMSO-ctrl. DMSO-ctrl. 10 100 1000 10000 0 250 500 750 1000 Cell membrane disrup�on (14h a�er OMX-0407 treatment) OMX-407 [nM] Cytotox red increase [%] 0 ng/ml TNF 1 ng/ml TNF DMSO-ctrl. DMSO-ctrl. MC38 murine CRC 10 100 1000 10000 0 5×105 1×106 1.5×106 2×106 2.5×106 Caspase 3/7 cleavage (8h a�er OMX-0407 treatment) OMX-407 [nM] Caspase-3 /7 cleavage (RLU) 0.8 ng/ml IgG-ctrl. mAb 0.8 ng/ml αDR5-mAb DMSO-ctrl. DMSO-ctrl. 10 20 40 80 160 0 100 200 300 400 500 600 700 Phospha�dylserine expression (14h a�er OMX-0407 treatment) OMX-407 [nM] Annexin V increase [%] 0.8 ng/ml IgG-ctrl. mAb 0.8 ng/ml αDR5-mAb DMSO-ctrl. DMSO-ctrl. 10 20 40 80 160 0 300 600 900 1200 1500 Cell membrane disrup�on (14h a�er OMX-0407 treatment) OMX-407 [nM] Cytotox red increase [%] 0.8 ng/ml IgG-ctrl. mAb 0.8 ng/ml αDR5-mAb DMSO-ctrl. DMSO-ctrl. EMT6 murine TNBC 10 100 1000 10000 0 2.5×105 5.0×105 7.5×105 1.0×106 1.3×106 Caspase 3/7 cleavage (8h a�er OMX-0407 treatment) OMX-407 [nM] Caspase-3 /7 cleavage (RLU) 0 ng/mL TRAIL 10 ng/mL TRAIL DMSO-ctrl. DMSO-ctrl. 10 100 1000 10000 0 2000 4000 6000 8000 Phospha�dylserine expression (24h a�er OMX-0407 treatment) OMX-407 [nM] Annexin V increase [%] 0 ng/ml TRAIL 10 ng/mL TRAIL DMSO-ctrl. DMSO-ctrl. 10 100 1000 10000 0 1000 2000 3000 4000 Cell membrane disrup�on (24h a�er OMX-0407 treatment) OMX-407 [nM] Cytotox red increase [%] 0 ng/mL TRAIL 10 ng/mL TRAIL DMSO-ctrl. DMSO-ctrl. A375 human Melanoma FIGURE II Murine colorectal carcinoma cell line MC38, murine triple negative breast cancer cell line EMT6 and human melanoma tumor cell line A375 were treated with 1 ng/ml TNF, 0.8 ng/ml murine death receptor 5 targeting antibody (clone MD5-1), 10 ng/ml complexed TRAIL or appropriate controls in combination with OMX-0407 and an associated DMSO-control. The onset of tumor cell death was monitored 8 hours after treatment initiation by cleavage of effector caspases 3/7, while the increase in extracellular phosphatidylserine expression was analyzed by annexin V and cell membrane disruption by cytotox red staining 24 hours after therapy initiation. 10 100 1000 10000 0 20 40 60 80 100 120 NFκB inhibi�on (7h a�er OMX-0407 treatment) OMX-407 [nM] NF-kB ac�vity [%] 0 ng/ml TNF 10 ng/ml TNF DMSO-ctrl. DM SO-ctrl.DMSO ctrl. 10 200 1500 0 50 100 150 WesternBlot - MC38 tumor cells phospho-HDAC4/5 OMX-407 [nM] % rel. pHDAC4/5 expression compared to DMSO - control 30 min 4 h DMSO-ctrl. 10 nM OMX-0407 200 nM OMX-0407 1500 nM OMX-0407 140 kDa: pHDAC4/5 (Ser246/Ser259) 124 kDa: pHDAC7 (Ser155) FIGURE III MC38wt. (A) and MC38-NF-κB luc reporter tumor cells (B) were treated in vitro with 10 ng/ml TNF or corresponding controls in combination with OMX-0407 and an associated DMSO-control. Tumor cells were lysed 30 min or 4h after treatment start and analyzed for phosphorylated histone deacetylase 4/5/7 by Western Blot. (A) Representative Blots are shown for 4 hour treatment. Phosphorylated HDAC4/5 was quantified and normalized to total protein load. Modulation of pHDAC4/5 level compared to DMSO control are shown. B) MC38-NF-κB luc reporter activity was monitored after 7h treatment with OMX-0407 or an associated DMSO-control. A) Pharmacokinetics of OMX-0407 in individual tissues after single OMX-0407 administration Plasma Tumor Skin B) Prolonged downregulation of NF-κB activity with high-dose OMX-0407 0.5 1 2 4 8 16 32 64 0 50 100 150 200 Time [h] % NF-κB ac�vity [photons/s] rel. to vehicle ctrl. Vehicle 5 mg/kg 50 mg/kg Baseline FIGURE IV MC38-NF-κB luc reporter tumor cells were subcutaneously implanted in C57Bl6/N mice, randomized at an average tumor volume of 400mm³ and treated with a single dose of 5 mg/kg or 50 mg/kg OMX-0407 or appropriate vehicle control by oral gavage. A) OMX-0407 concentration was analyzed by LC-MS in plasma, skin and tumor tissue at the indicated time points. B) Transcriptional activity of the NF-κB luc reporter was monitored non-invasively by bioluminescence imaging (BLI) in eight animals per time point. Mean BLI-signal for the indicated time points is shown normalized to vehicle control. C) Representative example of BLI signals 8 hours after start of treatment of animals with vehicle and 50 mg/kg OMX-0407. D) MC38 tumor tissue was collected 8h after treatment initiation. Tumor tissue lysates were analyzed ex vivo by Western Blot for phosphorylation of HDAC4/5/7 and NF-κB subunit p65 as well as level of cleaved caspase 3. Representative Blots of the 5 mg/kg and 50 mg/kg therapy group are shown for each analyte. OMX-0407 a potent inhibitor of SIK3, enhances death receptor mediated apoptosis in tumor cell lines of various indications  → OMX-0407 is a single-digit nanomolar inhibitor of salt-inducible kinase 3  → OMX-0407 enhances apoptosis in murine and human tumor cell lines upon TNF- or TRAIL treatment in a dose-dependent manner A) OMX-0407 treatment downregulates phosphorylation of histone deacetylase 4/5 in MC38 tumor cells B) OMX-0407 reduces TNF-induced NF-κB activity in NF-κB luc reporter cells In vivo pharmacokinetics of OMX-0407 determines dose-dependent pharmacodynamic abrogation of the SIK3 - NF-κB pathway activity in MC38 tumors  → OMX-0407 demonstrates dose dependent accumulation in tumor and peripheral tissues upon oral application  → OMX-0407 prevents intratumoral, transcriptional activity of NF-κB in an MC38-NF-κB reporter model associated to its pharmacokinetics  → OMX-0407 prohibits phosphorylation of HDAC4/5 and NF-κB subunit p65 and triggers tumor cell death Vehicle-ctrl.50 mg/kg OMX-0407 C) Non-invasive monitoring of NF-κB activity by in vivo Bioluminescence imaging 5 mg/kg OMX-0407 50 mg/kg OMX-0407 140 kDa: pHDAC4/5 (Ser246/Ser259) 124 kDa: pHDAC7 (Ser155) 65 kDa: pNF-κB p65 (Ser536) pHDAC4/5/7 pNF-κB p65 cleaved Caspase-3 17/19 kDa: cleaved casp.3 (ASP175) D) Phosphorylation of HDAC4/5 and NF-κB subunit p65 is inhibited by OMX-0407 in MC38 tumor tissue OMX-0407 repolarizes the tumor microenvironment and shows strong anti-tumor efficacy in monotherapy and in combination with PD-1 blockade  → OMX-0407 demonstrates highly significant anti-tumor efficacy and prolonged survival in monotherapy in the colorectal carcinoma model MC38  → OMX-0407 repolarizes the tumor microenvironment by reducing the number of regulatory T cells (Treg) and immunosuppressive myeloid cells and potentiating the cytotoxic T lymphocyte (CTL) activity as well as promoting the CTL to Treg ratio  → Combination treatment of OMX-0407 together with anti-PD-1 exploites the full potential of a repolarized anti-tumor immune environment 0 20 40 60 80 100% of CD45+cells 0.05 0.03 0 5 10 15 20 % of CD45+cells <0.001 0.003 0 20 40 60 % of CD45+cells <0.001 <0.007 0 20 40 60 ra�o 0.003 0 2 4 6 8% of CD45+cells <0.001 0 5 10 15 20 % of CD45+cells <0.007 CD8⁺ T cells CD25 ⁺ CTLs GrzB ⁺ CTLs (CD8⁺ CD25⁺) (CD8⁺ GrzB⁺) CTL / Treg ra�o M2-TAM M2-like-TAM (CD206⁺ MHCII-) (CD206⁺ MHCII⁺) A) Strong anti-tumor efficacy and prolonged survival by OMX-0407 therapy B) Repolarization toward an anti-tumor immune environment FIGURE V MC38 tumor cells were subcutaneously implanted in C57Bl6/N mice, randomized at an average tumor volume of 100 mm³ and tumor bearing mice were treated twice daily with 25 mg/kg OMX-0407 by oral gavage and twice weekly, intraperitoneal with 10 mg/kg anti-PD-1 mAb (clone: RMP1-14) or corresponding controls. A) Average tumor growth kinetics and overall survival. B) Tumor infiltrating cytotoxic T cells (CTL), CTL to regulatory T cell ratio, tumor associated M2-macrohages (M2-TAM) and M2-like TAM were analyzed by flow cytometry. 0 500 1000 1500 day a�er start of therapy Tumor volume (mm³) mean ± SEM 0 50 100 day a�er start of therapy % Survival 10 ml/kg vehicle + 10 mg/kg mIgG1e ctrl. 10 ml/kg vehicle + 10 mg/kg αPD-1 25 mg/kg OMX-0407 + 10 mg/kg mIgG1e ctrl. 25 mg/kg OMX-0407 + 10 mg/kg αPD-1 0 10 0 2020 30 40 60 OS = 22 d OS = 36 d OS = 43 d OS = 50 d 5 9% TGI 73% TGI 81% TGI 0 500 1000 1500 2000 2500 day a�er start of therapy tumor volume (mm³) 0 50 100 day a�er start of therapy % Survival mean ± SEM OS = 23 d OS = 30 d OS = 22 d OS = 34 d 10 ml/kg vehicle + 10 mg/kg mIgG1e ctrl. 10 ml/kg vehicle + 10 mg/kg αPD-1 12.5 mg/kg OMX-0407 + 10 mg/kg mIgG1e ctrl. 12.5 mg/kg OMX-0407 + 10 mg/kg αPD-1 0 10 0 2020 30 40 60-10 OMX-0407 acts synergistically with PD-1 blockade in immune checkpoint-resistant breast and lung cancer models  → Combination therapy of OMX-0407 and PD-1 blockade acts synergistically in the immune-excluded breast cancer model EMT6 and the immune checkpoint-resistant lung squamous cell carcinoma model KLN205  → Complete tumor regression and extension of overall survival were achieved in EMT6 tumors and significantly delayed tumor outgrowth in KLN205 lung cancer FIGURE VI EMT6 tumor cells were implanted into mammary fat pad in BALB/c mice and randomized at an average tumor volume of 65mm³. KLN205 tumor cells were subcutaneously implanted in DBA/2 mice and randomized at an average tumor volume of 100mm³. Both animal models were treated twice daily by oral gavage with the indicated doses of OMX-0407 and twice weekly, intraperitoneal with 10 mg/kg anti-PD-1 mAb (clone: RMP1-14) or corresponding controls. A) Average tumor growth kinetics and overall survival monitored for the EMT6 tumor model after treatment start. Two out of ten animals demonstrated complete tumor regression. B) Average tumor growth kinetics for the KLN205 tumor model upon initiation of therapy. A) Tumor growth inhibition/remission in the immune-excluded EMT6 breast cancer model B) Tumor growth inhibition in the anti-PD-1-resistant KLN205 lung squamous cell carcinoma model AACR 2022 #3708 TNFR1 Death complex e ector caspases 3/7 TNF T cell enhanced Apoptosis Caspase 8 CD95 & DR4/5 pro-Caspase-8 HDAC4 LKB1 NFkB SIK3 IkB NFkB HDAC4 nuclear reten on no transcription T cell OMX-0407 0 20 40 60 80 10 100 1000 10000 Time a�er drug administra�on [h] OMX-0407 [nM] 5 mg/kg LLQ 50 mg/kg 0 20 40 60 80 10 100 1000 10000 Time a�er drug administra�on [h] OMX-0407 [nmol/mg protein] 5 mg/kg 50 mg/kg LLQ 0 20 40 60 80 10 100 1000 10000 Time a�er drug administra�on [h] OMX-0407 [nmol/mg protein] 5 mg/kg 50 mg/kg LLQ 0 250 500 750 1000 1250 1500 1750 2000 day a�er start of therapy Tumor volume (mm 3) 54% TGI 32% TGI 10 ml/kg vehicle + 10 mg/kg mIgG1e ctrl. 10 ml/kg vehicle + 10 mg/kg αPD-1 25 mg/kg OMX-0407 + 10 mg/kg mIgG1e ctrl. 25 mg/kg OMX-0407 + 10 mg/kg αPD-1 mean ± SEM 0 10 20 30 40

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