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The Chk1 inhibitor, SRA737, demonstrates chemical synthetic lethality with replication stress-inducing agents, including novel low-dose gemcitabine, in preclinical models of cancer

Sierra Oncology, Inc., Vancouver, BC, Canada. Ryan J. Hansen, Bryan Strouse, Gregg Smith, Christian Hassig.

Translational Medicine Partners, Inc., San Diego, CA, USA. Kenna Anderes

Rationale • Replication Stress (RS) results in aberrant replication origin firing and the stalling of DNA replication forks, which has the potential to lead to DNA damage, genomic instability and cell death.

• Depending on genetic and other factors, tumor cells exhibit intrinsic RS to various extents. Low levels of RS contribute to genomic instability, an enabling hallmark of cancer, whereas high levels may be cytotoxic.

• Chemotherapeutic agents are typically used at maximally tolerated doses to induce cytotoxicity by causing cell death or preventing cell growth. While lower, sub-therapeutic doses of some chemotherapeutic agents including nucleoside analogs, DNA cross linkers and topoisomerase inhibitors, are not inherently cytotoxic, they are effective extrinsic inducers of RS through a range of mechanisms.

• The DNA damage response (DDR) checkpoint kinase 1, Chk1, is an essential effector of the cellular RS response. Consequently, tumor cells become highly reliant on Chk1 to manage RS in order to maintain proliferation and survival2,3. Therefore, RS inducing chemotherapies prime tumor cells to be highly sensitive to the anti-tumor effects of Chk1 inhibition via chemical synthetic lethality.

• Here we evaluated the potent, selective oral Chk1 inhibitor, SRA7374, for its potential to synergize with several clinically used chemotherapeutic inducers of RS to kill tumor cells in vitro and in vivo.

Figure 1. Replication stress-inducing chemotherapy, such as gemcitabine, activates a Chk1-dependent RS response. Subsequent inhibition of Chk1 by SRA737 results in excessive RS, replication catastrophe and tumor cell death.

Figure 2. SRA737 strongly synergized with all RS-inducing chemotherapies tested across multiple indications, with combination indices (CI) indicative of high synergy (CI < 0.7); strongest synergy was observed with gemcitabine. SRA737 was combined in a dose-optimized combination matrix with the indicated chemotherapy and evaluated across a 15 cell line panel including: 5637, TCCSUP and J82 (Bladder); HT-29 and SW620 (Colon); CAL-27 and FaDu (Head and neck squamous carcinoma); Calu-6 and NCI-H520 (Lung); KURAMOCHI, OV90 and OVCAR-3 (Ovarian); MIA PaCa-2, Panc 03.27 and SNU-324 (Pancreatic). CI scores were calculated according to the method of Chou & Talalay5 across the combination dose matrix. The mean and range best CI scores and number of cell lines with a synergistic CI are reported below. CI scores < 0.7 indicate synergy, CI scores between 0.7 and 1.3 indicate additivity, and CI scores > 1.3 are antagonistic. For reference, CIs have been previously reported to range from 0.44 – 1.26 when combining gemcitabine with carboplatin in 5637 bladder cells depending on dose and order of addition 6.

Figure 3. SRA737 synergizes with type I and type II topoisomerase inhibitors to inhibit growth and potentiate cytotoxicity in tumor cell lines. Type I and type II topoisomerase inhibitors disrupt DNA topology and generate covalent DNA adducts that interfere with the DNA replication machinery and result in stalled replication forks and RS. These effects are expected to synergize with Chk1 inhibition to induce excessive RS, DNA damage and cell death, even at lower concentrations of topoisomerase inhibitors. Combination treatment with type I and type II topoisomerase inhibitors, either SN-38 (96 h) or etoposide (96 h), respectively, and SRA737 (72 h) in various cell lines leads to potentiation of growth inhibition. Similar to its effects with other agents, SRA737 combined with either SN-38 or etoposide resulted in cytotoxic activity at lower doses of either topoisomerase inhibitor (in KURAMOCHI and OVCAR-3 cells) or when no cytotoxic activity was observed with either SN-38 or etoposide alone (in MIA PaCa-2 and CAL-27 cells). HGSOC, high-grade serous ovarian cancer; PDAC, pancreatic ductal adenocarcinoma; HNSCC, head and neck squamous cell carcinoma.

Figure 5. Gemcitabine synergizes with SRA737 in bladder carcinoma cell lines and patient tumor-derived 3D cultures. Combination treatment in three bladder cell lines leads to increased growth inhibition, as evidenced at lower doses of gemcitabine (4 or 12 nM) (A). Potentiation of gemcitabine-induced growth inhibition (as determined by Alamar Blue 72 h after addition of gemcitabine and 66 h after SRA737 treatment) in primary bladder cancer patient 3D cultures, demonstrating a substantial increase in potency of the combination (B).

Figure 6. SRA737 plus gemcitabine inhibits tumor growth and extends survival in a gemcitabine-insensitive PDX model of bladder cancer. Mice bearing patient derived xenograft (PDX) tumors were treated with vehicle or gemcitabine (100 mg/kg, IP; green arrows) and vehicle or SRA737 (100 mg/kg, PO; blue arrows) when initial tumor volumes were approximately 100 mm3. The gemcitabine / SRA737 combination inhibited tumor growth (tumor growth inhibition = 64% of vehicle control). Monotherapy SRA737 resulted in no tumor growth inhibition (data not shown). ** = Log-rank (Mantel-Cox) test Gem(100)+SRA737(100) vs Gem(100) (P=0.0109) or Vehicle (P=0.0162).

Figure 7. SRA737 synergizes with sub-therapeutic doses of gemcitabine in vivo. Mice bearing subcutaneous colorectal HT-29 or osteosarcoma SJSA-1 tumors were treated with vehicle or gemcitabine (IV; green arrows) plus vehicle or SRA737 (PO; blue arrows) at indicated doses once initial tumor volumes were approximately 100 mm3. HT-29 tumor growth inhibition was significantly better in an SRA737 dose-related fashion when combined with gemcitabine (40 mg/kg) vs. gemcitabine administered alone at 80 mg/kg. Similarly, the SRA737 plus gemcitabine combination was significantly more efficacious than gemcitabine alone at inhibiting SJSA-1 xenograft tumor growth. In both HT-29 and SJSA-1 tumor models, monotherapy SRA737 was not efficacious at the comparable dose and schedule (data not shown).

Figure 8. SRA737 combined with a sub-therapeutic dose of gemcitabine elevates RS markers in vivo. HT-29 tumor bearing mice were treated with a single IV dose of gemcitabine (40 mg/kg) followed 24 h later by a single dose of SRA737 or vehicle prior to tumor collection 12 h post SRA737 or vehicle dose (A). Tumor lysates prepared from two to three animals per treatment group were analyzed by Western blot (B). Dose concordant in vivo inhibition of Chk1 by SRA737 (p-Chk1 (S296) autophosphorylation site) increases markers of replication fork stalling (ATR phosphorylation sites on Chk1 (S317 and S345) and p-RPA32 (S33)) and fork collapse (γ-H2A.X) induced by a single low dose of gemcitabine. Total protein levels for H2A.X and RPA32 were unchanged (data not shown).

(A) Under conditions of RS, such as those induced by low-dose gemcitabine, dysregulation of the DNA replication machinery, slowing or stalling of polymerases at replication forks, and depletion of of deoxyribonucleotide triphosphate (dNTP) supply can result in extended stretches of fragile single stranded DNA (ssDNA) coated with Replication Protein A (RPA).

(B) Persistent lengths of RPA-bound ssDNA are recognized by ataxia telangiectasia and Rad3-related (ATR) kinase, contributing to the activation of the cellular RS response, which involves phosphorylation of key substrates including RPA on S33 (p-RPA32 (S33)). Activated ATR also phosphorylates Chk1 at S317 and S345 (p-Chk1 (S317) and p-Chk1 (S345)) leading to Chk1 activation and subsequent autophosphorylation at S296 (p-Chk1 (S296)). As an essential effector of the response to RS, activated Chk1 stabilizes stalled replication forks, abrogates new origin firing, arrests the cell cycle, and fosters DNA repair.

(C) Inhibition of Chk1 by SRA737, in the presence of RS, results in excessive fork stalling and destabilization, leading to fork collapse, DNA double strand break damage and a reduced propensity to repair the damaged DNA.

For more information, email info@sierraoncology.com or visit www.sierraoncology.com

Acknowledgements

• SRA737 was discovered and initially developed by the Cancer Research UK (CRUK) Cancer Therapeutics Unit at the Institute of Cancer Research (ICR).

• The authors would also like to thank Eric J. Brown, Karlene Cimprich, Alan D. D’Andrea, Johann de Bono, Alan R. Eastman, Michelle D. Garrett, Thomas Helleday and Leonard Post for their helpful discussions regarding these experiments.

References:

1. Toledo, L., Neelsen, K. J. & Lukas, J. Replication Catastrophe : When a Checkpoint Fails because of Exhaustion. Mol. Cell 66, 735–749 (2017).

2. Koh, S. B. et al. CHK1 inhibition synergizes with gemcitabine initially by destabilizing the DNA replication apparatus. Cancer Res. 75, 3583–3595 (2015).

3. McNeely, S., Beckmann, R. & Bence Lin, A. K. CHEK again: Revisiting the development of CHK1 inhibitors for cancer therapy. Pharmacol. Ther. 142, 1–10 (2014).

4. Walton, M. I., et al. The clinical development candidate CCT245737 is an orally active CHK1 inhibitor with preclinical activity in RAS mutant NSCLC and Eµ-MYC driven B-cell lymphoma. Oncotarget 7, 2329–42 (2016).

5. Chou. T. & Talalay P. Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul. 22, 27-55 (1984).

6. Wang, S., et al. Analysis of the cytotoxic activity of the carboplatin and gemcitabine combination. Anticancer Res. 30(11), 4573-8 (2010).

SRA737 Combined with RS-Inducing Chemotherapies SRA737 Combined with Gemcitabine in Bladder Carcinoma

Conclusions • Replication stress, comprising of aberrant replication origin firing and stalling of DNA replication forks, leads to DNA damage, genomic instability and cell death. Tumor cells exhibit intrinsic RS to various extents.

• Gemcitabine, as well as several other chemotherapeutic drugs including DNA cross-linkers and topoisomerase inhibitors, can cause deoxyribonucleotide triphosphate (dNTP) depletion and/or DNA damage resulting in elevated extrinsic RS1.

• The DNA damage response (DDR) checkpoint kinase 1, Chk1, is essential for managing RS by stabilizing replication forks, abrogating origin firing, pausing the cell cycle and mediating DNA repair. Consequently, tumor cells become highly reliant on Chk1 to manage RS and its downstream consequences in order to survive and continue to proliferate 2,3.

• Here we demonstrate that the potent, selective, oral Chk1 inhibitor, SRA737, strongly synergizes with a range of chemotherapeutic agents that induce RS, resulting in tumor cell death in vitro and in vivo via chemical synthetic lethality.

• The combination of SRA737 with a range of RS-inducing agents was found to be highly synergistic (CI < 0.7) in a panel of 15 cell lines of diverse tissue lineages, with the strongest synergy observed with gemcitabine (Figure 2).

• The combination of SRA737 with DNA cross-linking agents or a nucleoside analog increases cellular RS markers (e.g. pChk1 (S317/345), p-RPA (S33) and γ-H2A.X) (Figure 4).

• Profound synergy between SRA737 and gemcitabine was also observed in several bladder cancer cell lines (5637, TCCSUP and J82) as well as in human patient-derived bladder cancer 3D cultures – further supporting the clinical development of this promising RS-inducing combination (Figure 5).

• Significant anti-tumor activity (tumor growth inhibition = 64%) and increased survival (vs. control) were observed when SRA737 and gemcitabine were dosed in combination, in a highly aggressive gemcitabine resistant bladder carcinoma PDX model (Figure 6). These findings suggest that the combination of SRA737 and gemcitabine may be efficacious in gemcitabine-resistant clinical settings.

• Strikingly, anti-tumor activity (tumor growth inhibition > 70%) was observed when SRA737 was combined with a sub-therapeutic dose of gemcitabine (40 mg/kg) in xenograft models of colorectal adenocarcinoma and osteosarcoma (Figure 7). This combination was shown to increase RS markers by three to five-fold over the change noted with gemcitabine alone (Figure 8). These findings support the broader application of this unique combination in tumor indications where gemcitabine is not standard of care, and the development of SRA737 in combination with low, sub-therapeutic doses of gemcitabine.

• SRA737 is currently being evaluated in two Phase I clinical trials investigating the safety, tolerability and pharmacokinetics of monotherapy SRA737 (NCT02797964) and in novel combination with low-dose gemcitabine (NCT02797977) in patients with advanced cancer.

SRA737 Combined with Sub-Therapeutic Gemcitabine

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Vehicle controlGemcitabine (100 mg/kg) Q7DGem (100) + SRA737 (100)

Figure 4. SRA737 synergizes with sub-therapeutic concentrations of standard of care chemotherapeutics to increase tumor cell death and induce markers of replication stress. Combination treatment of gemcitabine (A), cisplatin (C) or mitomycin C (E), even at low, sub-therapeutic concentrations of each agent, with SRA737 leads to increased growth inhibition. Inhibition of Chk1 by SRA737 (p-Chk1 (S296) autophosphorylation site) dramatically increases markers of replication fork stalling (ATR phosphorylation sites on Chk1 (S317 and S345) and p-RPA32 (S33)) and fork collapse (γ-H2A.X) induced by low concentrations of gemcitabine (B), cisplatin (D) or mitomycin C (F). Total protein levels for H2A.X and RPA32 were unchanged (not shown).

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