Figure 4. SRA737 elevates RS markers and induces DNA damage in vivo. OVCAR3 tumor bearing mice were treated with a single dose of SRA737 or vehicle and the tumors were collected 12 h later. Tumor lysates prepared from three animals per treatment group were analyzed by Western blot. Dose concordant increases in markers of RS / fork stalling (ATR phosphorylation sites on Chk1 (S317 and S345) and p-RPA32 (S33)) and fork collapse (γ-H2AX) were observed in vivo. Total protein levels for H2AX and RPA32 were unchanged (data not shown). These results suggest that selective inhibition of Chk1 in CCNE1amp tumors with high intrinsic RS results in excessive RS and extensive DNA damage and genomic instability that is incompatible with tumor cell survival.

The Novel Oral Chk1 Inhibitor, SRA737, is Active in Both PARP Inhibitor Resistant and CCNE1 Amplified High Grade Serous Ovarian Cancers Abstract #LB-265

Haineng Xu1, Sergey Medvedev1, Ashka Pandya1, Hyoung Kim1, Yasuto Kinose1, Eric Brown2, Ryan J. Hansen3, Bryan Strouse3, Snezana Milutinovic3, Christian Hassig3, Fiona Simpkins1

Departments of Obstetrics and Gynecology1, Cancer Biology2, University of Pennsylvania, Philadelphia, PA; Sierra Oncology, Inc.3, Vancouver, BC, Canada.

Rationale Rationale

• Approximately 20% of high grade serous ovarian cancers (HGSOC) harbor CCNE1 gene amplification (CCNE1amp). CCNE1 encodes the oncogene cyclin E. These tumors show intrinsic resistance to PARPi and frequently are or become resistant to platinum therapy. • CCNE1amp is known to increase replication stress (RS) and genomic instability, leading to increased reliance on Checkpoint kinase 1 (Chk1). As an essential effector of the cellular response to RS, activated Chk1 stabilizes stalled replication forks, abrogates new origin firing, temporarily arrests the cell cycle, and fosters DNA repair to avoid replication- and mitotic catastrophe-induced cell death. • Here, we investigated the in vitro and in vivo anti-tumor activity of the potent, highly selective, orally bioavailable small molecule inhibitor of Chk1, SRA737, in CCNE1amp HGSOC models. • We hypothesized that intrinsically elevated RS, as a result of cyclin E overexpression, would predispose these tumors to Chk1i-induced catastrophic genomic instability following treatment with SRA737.

Chk1 Inhibition Results in Catastrophic Dysregulation of Replication, Leading to Cancer Cell Death

Chk1 Enables Survival of Cancer Cells by Managing High Levels of RS

Figure 2. SRA737 is active as monotherapy in a CCNE1amp;TP53 (R248Q) mutant cell line, resulting in RS and DNA double strand breaks. OVCAR3 cells (CCNE1amp; TP53 (R248Q)) were treated with vehicle or SRA737 (0.1 or 0.2 µM) for 12 days. Crystal violet staining was quantified using ImageJ and percent of control was determined (A). OVCAR3 cells were treated with vehicle or SRA737 (0.2 µM) for 24 h prior to cell lysis and Western blot analysis for indicated markers (B). Persistent lengths of RPA-bound ssDNA, a hallmark of RS, 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 (p-RPA32). Activated ATR also phosphorylates Chk1 at S317 and S345, leading to Chk1 activation. SRA737 treatment resulted in an increase in p-Chk1 (S345). SRA737 also led to an increase in γ-H2AX, an indicator of DNA double strand breaks and replication fork collapse. FACS analysis for DNA bound p-RPA32 (C) and γ-H2AX positivity (D) in OVCAR3 cells following 24 h of treatment with vehicle or SRA737 (1 µM). Taken together, data in OVCAR3 cells show that monotherapy SRA737 increases RS and DNA double strand breaks within 24 h, resulting in a reduced ability of these cells to form colonies during 12 days of treatment with SRA737.

Figure 1. Cancer Cells are Dependent on Chk1 to Manage High Levels of RS and Survive.

Figure 3. SRA737 is active as monotherapy in CCNE1amp;TP53 (R248Q) HGSOC xenografts. Mice bearing subcutaneous ovarian OVCAR3 tumors were treated with SRA737 (100 mg/kg QD, PO) or vehicle for three cycles of 5 days on/ 2 days off. Significant tumor growth inhibition (60%) was observed to study termination, 6 days after last treatment. These results suggest CCNE1amp ovarian tumors with high intrinsic RS are heavily reliant on Chk1 activity with consequent sensitivity to SRA737 therapy.

Figure 7. SRA737 is synergistic with PARPi in HRD HGSOC cancer cells. HRD ovarian cancer cells (JHOS-4 [A] and PEO1 [B]) were exposed to increasing concentrations of SRA737 (0 – 0.5 µM), PARPi (0.05 – 0.5 µM) or combinations of the two agents continuously for 12 days. Tumor cell colonies were stained with crystal violet and quantified using ImageJ, and the coefficient of drug interaction (CDI) was calculated. The combination of SRA737 with PARPi was synergistic in decreasing colony formation in HRD cells (JHOS-4, best CDI=0.45; PEO1, CDI=0.30).These data suggest that SRA737 exerts single-agent activity in HRD cell lines, and also synergizes with PARPi in this setting. Note Y-axis is log scale.

Figure 8. SRA737 is synergistic with PARPi in HGSOC that acquired PARPi resistance. PARPi-resistant cells (PEO1-PR [A] and PEO4 [B]) were exposed to increasing concentrations of SRA737 (0 – 0.2 µM), PARPi (0.5 or 1 µM) or combinations of the two agents continuously for 12 days and analyzed as in Figure 7. These cells exhibit marginal response to PARPi, which confirms their PARPi-resistance and potential restoration of HRR. Conversely, these cells demonstrated marked sensitivity to SRA737 monotherapy. In addition, consistent with HRD cell lines, the combination of SRA737 with PARPi was synergistic in decreasing the colony forming ability of these PARPi-resistant cells (PEO1-PR, CDI=0.11; PEO4, CDI=0.08).

Figure 9. SRA737 is active in combination with PARPi in HGSOC PDX. Orthotopically implanted HGSOC tumors collected from a platinum resistant patient were treated with PARPi until PARPi-resistance developed (24 weeks) and were then harvested. After a second implantation, tumors reaching 50 mm3 were grown in the presence of PARPi (50-100 mg/kg; most 75 mg/kg; QD) until tumors grew to 100 mm3. At that time, mice either remained on PARPi (75 mg/kg; QD) or were switched to SRA737 (75 mg/kg; 5 days on / 2 days off) alone or in combination with PARPi. Preliminary evidence in this PDX model demonstrates tumor growth inhibition by SRA737 in combination with PARPi. Consistent with in vitro results, SRA737 is active as a monotherapy at higher doses in this model. All treatments were well tolerated (data not shown). These results support the potential clinical utility of the combination of SRA737 plus PARPi to overcome acquired PARPi resistance in patients.

SRA737 Monotherapy in CCNE1amp High Grade Serous Ovarian Cancers

Conclusions

SRA737 + PARPi in HRR deficient and Acquired PARPi-resistant High Grade Serous Ovarian Cancers

• HRR deficient (HRD) HGSOC are initially sensitive to PARPi, but drug resistance ultimately emerges, frequently involving genetic reversion of BRCA mutated genes and partial restoration of HRR.

• Data presented here demonstrates that Chk1 inhibition by SRA737 in combination with PARPi was synergistic in decreasing colony formation in HRD and acquired PARPi-resistant cell models. Consistent with Chk1’s role in HRR, SRA737 was active as a single agent in HRD cell lines.

• SRA737 demonstrated single agent activity in acquired PARPi-resistant cells, as well as in combination with PARPi.

• CCNE1amp HGSOC represent a subset of patients (~ 20%) with high replication stress (RS) that show intrinsic resistance to PARPi and platinum treatments.

• Data presented here demonstrates that Chk1 inhibition by SRA737 increases the level of RS and DNA double strand breaks, providing evidence that excessive genomic instability results in subsequent cell death and tumor regression in CCNE1amp ovarian cancer models.

Conclusions

A

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VehicleSRA737 (0.2 μM)SRA737 (0.5 μM)

Figure 5. SRA737 is active as a monotherapy in orthotopic HGSOC CCNE1amp; TP53 (R342X) PDX. HGSOC tumors collected from a platinum resistant patient were orthotopically implanted on the fallopian tubes of recipient mice. Once tumors reached ~100 mm3 by ultrasound measurements, mice were assigned to vehicle, SRA737 (50 mg/kg; PO, 5 days on / 2 days off) or PARPi (50 mg/kg; PO). SRA737 at clinically achievable human equivalent doses administered in a 5 on / 2 off schedule resulted in significant tumor growth delay (A) and a profound survival benefit (B) as compared to vehicle or PARPi alone. Both drugs were well tolerated over the extended duration of this study. The weak efficacy of PARPi in this model is consistent with the lack of clinical efficacy of PARPi in this highly aggressive subset of HGSOC.

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Figure 6. Rationale for Potential Synergy Between SRA737 + PARPi.

• Chk1’s role regulating replication stress and HRR facilitates several potential SRA737 + PARPi therapeutic scenarios via ‘chemical synthetic lethality’, the synergistic, contemporaneous inhibition of both Chk1 and PARP.

• Mutations in certain genes (e.g. BRCA1/2) impair replication fork stability and/or HRR (HRD settings). Chk1 inhibition by SRA737 increases replication stress, destabilizes replication forks, and impairs HRR.

• Partial restoration of HRR, e.g. reversion mutations in BRCA1/2, results in acquired PARPi-resistance, which may be overcome by Chk1 inhibition with SRA737.

• Consequently, SRA737 combination therapy with PARPi may (i) deepen responses in HRD tumors; (ii) overcome resistance following PARPi-therapy.

RS increases genomic instability

Oncogenic driverse.g. Dysregulation of

replication, transcription/ replication collision

CCNE1

Defective DNA damage repaire.g. Single strand breaks,

double strand breaks

BRCA 1/2

Genomic InstabilityNormal Cell

Cancer Cell Replicates

Cell Death

Cancer cells are dependent on Chk1to manage high levels of RS and survive

Chk1regulates RS

Chk1

RS increases genomic instability

Genomic InstabilityNormal Cell

Chk1 inhibition results in catastrophic dysregulation of replication, leading to cancer cell death

Excessive genomic instability results in cancer cell death

RS increases genomic instability

Oncogenic driverse.g. Dysregulation of

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CCNE1

Defective DNA damage repaire.g. Single strand breaks,

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BRCA 1/2

Genomic InstabilityNormal Cell

Cancer Cell Replicates

Cell Death

Cancer cells are dependent on Chk1to manage high levels of RS and survive

Chk1regulates RS

Chk1

RS increases genomic instability

Genomic InstabilityNormal Cell

Chk1 inhibition results in catastrophic dysregulation of replication, leading to cancer cell death

Excessive genomic instability results in cancer cell death

• A distinct subgroup comprising approximately 50% of HGSOC have defective homologous recombination repair (HRR) genes. HRR deficient (HRD) HGSOC are initially sensitive to poly (ADP-ribose) polymerase inhibitors (PARPi) but drug resistance ultimately emerges, frequently involving genetic reversion of BRCA mutated genes and partial restoration of HRR. • Here, we investigated the in vitro and in vivo anti-tumor activity of the potent, highly selective, orally bioavailable small molecule inhibitor of Chk1, SRA737, in HRD and acquired PARPi-resistant HGSOC models. • For HRD tumors, we hypothesized that defective HRR (e.g. BRCA1/2 mutation) may elevate sensitivity to Chk1i, given the well-established role of Chk1 in HRR, as well as other aspects of RS response.

• Monotherapy SRA737 led to a highly reduced ability of OVCAR3 CCNE1amp / TP53 (R248Q) ovarian cells to form colonies. This was determined to be a downstream consequence of increased RS and DNA damage as measured by Western blot and flow cytometry.

• Consistent with in vitro activity, SRA737 significantly inhibited tumor growth in an OVCAR3 xenograft model. In an orthotopic PDX model established from a platinum-resistant CCNE1amp ovarian cancer patient, SRA737 demonstrated profound monotherapy activity and a profound survival benefit in this inherently PARPi-insensitive model.

• Analogous to PARPi, which first exhibited robust activity in patients harboring BRCA mutations, Chk1 inhibitors, such as SRA737, may prove effective in defined genetic backgrounds of high RS, such as CCNE1 amplification.

• The efficacy of SRA737 monotherapy is currently being investigated in CCNE1- and other RS-driven HGSOC in the extant Phase 1/2 clinical trial (NCT02797964).

• Furthermore, preliminary evidence in a HGSOC PDX model demonstrated synergistic tumor growth inhibitory activity of SRA737 in combination with PARPi.

• SRA737 is a potent, highly selective, orally bioavailable Chk1 inhibitor that demonstrates activity in PARPi-sensitive and acquired PARPi-resistant preclinical cancer models, warranting further development in these patient populations.

• A Phase 1b/2, multicenter, open-label, dose-ranging study to assess the safety, tolerability, pharmacokinetics, and preliminary antitumor activity of SRA737 given in combination with niraparib in subjects with metastatic castration-resistant prostate cancer (mCRPC) is being planned.

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Post-PARPi Resistant ‘Overcome Resistance’

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Indicators of replication fork stallingp-Chk1 S345

p-RPA32 S33

γ-H2AX Indicator of DSB / Replication fork collapse

GAPDH

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

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