Dongpo Cai

LKB1 modulates lung cancer differentiation and metastasis.

Germline mutation in serine/threonine kinase 11 (STK11, also called LKB1) results in Peutz–Jeghers syndrome, characterized by intestinal hamartomas and increased incidence of epithelial cancers. Although uncommon in most sporadic cancers, inactivating somatic mutations of LKB1 have been reported in primary human lung adenocarcinomas and derivative cell lines. Here we used a somatically activatable mutant Kras-driven model of mouse lung cancer to compare the role of Lkb1 to other tumour suppressors in lung cancer. Although Kras mutation cooperated with loss of p53 or Ink4a/Arf (also known as Cdkn2a) in this system, the strongest cooperation was seen with homozygous inactivation of Lkb1. Lkb1-deficient tumours demonstrated shorter latency, an expanded histological spectrum (adeno-, squamous and large-cell carcinoma) and more frequent metastasis compared to tumours lacking p53 or Ink4a/Arf. Pulmonary tumorigenesis was also accelerated by hemizygous inactivation of Lkb1. Consistent with these findings, inactivation of LKB1 was found in 34% and 19% of 144 analysed human lung adenocarcinomas and squamous cell carcinomas, respectively. Expression profiling in human lung cancer cell lines and mouse lung tumours identified a variety of metastasis-promoting genes, such as NEDD9, VEGFC and CD24, as targets of LKB1 repression in lung cancer. These studies establish LKB1 as a critical barrier to pulmonary tumorigenesis, controlling initiation, differentiation and metastasis.

Proapoptotic BH3-Only BCL-2 Family Protein BIM Connects Death Signaling from Epidermal Growth Factor Receptor Inhibition to the Mitochondrion

A subset of lung cancers expresses mutant forms of epidermal growth factor receptor (EGFR) that are constitutively activated. Cancers bearing activated EGFR can be effectively targeted with EGFR inhibitors such as erlotinib. However, the death-signaling pathways engaged after EGFR inhibition are poorly understood. Here, we show that death after inhibition of EGFR uses the mitochondrial, or intrinsic, pathway of cell death controlled by the BCL-2 family of proteins. BCL-2 inhibits cell death induced by erlotinib, but BCL-2-protected cells are thus rendered BCL-2-dependent and sensitive to the BCL-2 antagonist ABT-737. BH3 profiling reveals that mitochondrial BCL-2 is primed by death signals after EGFR inhibition in these cells. As this result implies, key death-signaling proteins of the BCL-2 family, including BIM, were found to be up-regulated after erlotinib treatment and intercepted by overexpressed BCL-2. BIM is induced by lung cancer cell lines that are sensitive to erlotinib but not by those resistant. Reduction of BIM by siRNA induces resistance to erlotinib. We show that EGFR activity is inhibited by erlotinib in H1650, a lung cancer cell line that bears a sensitizing EGFR mutation, but that H1650 is not killed. We identify the block in apoptosis in this cell line, and show that a novel form of erlotinib resistance is present, a block in BIM up-regulation downstream of EGFR inhibition. This finding has clear implications for overcoming resistance to erlotinib. Resistance to EGFR inhibition can be modulated by alterations in the intrinsic apoptotic pathway controlled by the BCL-2 family of proteins.

Combined depletion of cell cycle and transcriptional cyclin- dependent kinase activities induces apoptosis in cancer cells

Selective cyclin-dependent kinase (cdk) 2 inhibition is readily compensated. However, reduced cdk2 activity may have antiproliferative effects in concert with other family members. Here, inducible RNA interference was used to codeplete cdk2 and cdk1 from NCI-H1299 non-small cell lung cancer and U2OS osteosarcoma cells, and effects were compared with those mediated by depletion of either cdk alone. Depletion of cdk2 slowed G1 progression of NCI-H1299 cells and depletion of cdk1 slowed G2-M progression in both cell lines, with associated endoreduplication in U2OS cells. However, compared with the incomplete cell cycle blocks produced by individual depletion, combined depletion had substantial consequences, with G2-M arrest predominating in NCI-H1299 cells and apoptosis the primary outcome in U2OS cells. In U2OS cells, combined depletion affected RNA polymerase II expression and phosphorylation, causing decreased expression of the antiapoptotic proteins Mcl-1 and X-linked inhibitor of apoptosis (XIAP), effects usually mediated by inhibition of the transcriptional cdk9. These events do not occur after individual depletion of cdk2 and cdk1, suggesting that reduction of cdk2, cdk1, and RNA polymerase II activities all contribute to apoptosis in U2OS cells. The limited cell death induced by combined depletion in NCI-H1299 cells was significantly increased by codepletion of cdk9 or XIAP or by simultaneous treatment with the cdk9 inhibitor flavopiridol. These results show the potency of concomitant compromise of cell cycle and transcriptional cdk activities and may guide the selection of clinical drug candidates.

Cdk1 Participates in BRCA1-Dependent S Phase Checkpoint Control in Response to DNA Damage

Cdk2 and cdk1 are individually dispensable for cell-cycle progression in cancer cell lines because they are able to compensate for one another. However, shRNA-mediated depletion of cdk1 alone or small molecule cdk1 inhibition abrogated S phase cell-cycle arrest and the phosphorylation of a subset of ATR/ATM targets after DNA damage. Loss of DNA damage-induced checkpoint control was caused by a reduction in formation of BRCA1-containing foci. Mutation of BRCA1 at S1497 and S1189/S1191 resulted in loss of cdk1-mediated phosphorylation and also compromised formation of BRCA1-containing foci. Abrogation of checkpoint control after cdk1 depletion or inhibition in non-small-cell lung cancer cells sensitized them to DNA-damaging agents. Conversely, reduced cdk1 activity caused more potent G2/M arrest in nontransformed cells and antagonized the response to subsequent DNA damage. Cdk1 inhibition may therefore selectively sensitize BRCA1-proficient cancer cells to DNA-damaging treatments by disrupting BRCA1 function.

AZ703, an Imidazo[1,2-a]Pyridine Inhibitor of Cyclin-Dependent Kinases 1 and 2, Induces E2F-1-Dependent Apoptosis Enhanced by Depletion of Cyclin-Dependent Kinase 9

Preclinical studies were performed of a novel selective imidazopyridine cyclin-dependent kinase (cdk) inhibitor, AZ703. In vitro kinase assays showed that IC50 values for AZ703 against purified cyclin E/cdk2 and cyclin B/cdk1 were 34 and 29 nmol/L, respectively. In contrast, the IC50 against cdk4 was 10 micromol/L. AZ703 also inhibited cdk7 and cdk9 with IC50 values of 2.1 micromol/L and 521 nmol/L, respectively. Treatment of U2OS, NCI-H1299, and A549 cells for 24 hours resulted in growth arrest involving multiple cell cycle phases. At low drug concentrations (< 2 micromol/L), G2 arrest predominated, whereas at higher concentrations (> or = 2 micromol/L), S-G2 arrest was observed. When cells were synchronized in G1 by starvation and released into AZ703, a block in G1 occurred that was not evident in exponentially growing cells. Cell cycle arrest was associated with reduced phosphorylation of the retinoblastoma protein and p27(Kip1) at cdk2 phospho-sites. Following longer exposures, apoptosis was evident. Cells were further sensitized to AZ703 following recruitment to S phase by synchronization. Consistent with the inhibition of cdks during S and G2 that modulate the activity and stability of E2F-1, AZ703 treatment induced E2F-1 expression. In U2OS and NCI-H1299 cells engineered to inducibly express the dominant-negative mutant E2F-1 (1-374), expression of the mutant decreased AZ703-mediated apoptosis, indicating dependence on E2F-1 transcriptional targets. AZ703-induced apoptosis in NCI-H1299 cells was enhanced by small interfering RNA-mediated depletion of cdk9, which caused reduced levels of Mcl-1 and XIAP, suggesting that cdk2, cdk1, and cdk9 represent a rational subset of family members for drug targeting.

Phase I Safety, Pharmacokinetic, and Pharmacodynamic Study of the Poly(ADP-ribose) Polymerase (PARP) Inhibitor Veliparib (ABT-888) in Combination with Irinotecan in Patients with Advanced Solid Tumors

Purpose: PARP is essential for recognition and repair of DNA damage. In preclinical models, PARP inhibitors modulate topoisomerase I inhibitor–mediated DNA damage. This phase I study determined the MTD, dose-limiting toxicities (DLT), pharmacokinetics (PK), and pharmacodynamics (PD) of veliparib, an orally bioavailable PARP1/2 inhibitor, in combination with irinotecan. Experimental Design: Patients with advanced solid tumors were treated with 100 mg/m2 irinotecan on days 1 and 8 of a 21-day cycle. Twice-daily oral dosing of veliparib (10–50 mg) occurred on days 3 to 14 (cycle 1) and days −1 to 14 (subsequent cycles) followed by a 6-day rest. PK studies were conducted with both agents alone and in combination. Paired tumor biopsies were obtained after irinotecan alone and veliparib/irinotecan to evaluate PARP1/2 inhibition and explore DNA damage signals (nuclear γ-H2AX and pNBS1). Results: Thirty-five patients were treated. DLTs included fatigue, diarrhea, febrile neutropenia, and neutropenia. The MTD was 100 mg/m2 irinotecan (days 1 and 8) combined with veliparib 40 mg twice daily (days −1–14) on a 21-day cycle. Of 31 response-evaluable patients, there were six (19%) partial responses. Veliparib exhibited linear PK, and there were no apparent PK interactions between veliparib and irinotecan. At all dose levels, veliparib reduced tumor poly(ADP-ribose) (PAR) content in the presence of irinotecan. Several samples showed increases in γ-H2AX and pNBS1 after veliparib/irinotecan compared with irinotecan alone. Conclusions: Veliparib can be safely combined with irinotecan at doses that inhibit PARP catalytic activity. Preliminary antitumor activity justifies further evaluation of the combination. Clin Cancer Res; 22(13); 3227–37. ©2016 AACR.

Abstract CT325: Combination of the PARP inhibitor veliparib (ABT888) with irinotecan in patients with triple negative breast cancer: Preliminary activity and signature of response

Background: The nuclear enzyme PARP is essential in recognition and repair of DNA damage. Preclinical evidence suggests that PARP inhibitors work as sensitizing agents for DNA-damaging agents such as irinotecan. Veliparib is an orally bioavailable PARP 1 and 2 inhibitor. This expansion to a phase I study, which demonstrated veliparib reduces PAR levels in tumor after irinotecan exposure, was conducted to assess the safety, tolerability and preliminary anti-tumor activity of the combination of veliparib and irinotecan in triple negative breast cancer (TNBC) patients (pts), as well as to apply next generation sequencing technologies to define a signature of response. Methods: Pts were enrolled to two breast cancer cohorts: (1) TNBC, germline BRCA-mutant positive and (2) TNBC, non-BRCA mutated (wt). Eligibility included performance status 0-2; ≥ age 18; adequate bone marrow, hepatic and renal function. Cycles were 21 days. Irinotecan was given i.v. 100 mg/m2 over 90 min on Days 1 and 8. Twice daily (BID) oral dosing of 40 mg veliparib occurred Days 2-15 (Cycle 1) and Days 1-15 (subsequent cycles) followed by a 6-day rest. Tumor biopsies were collected at baseline, 4-6 hours after the first dose of irinotecan (day 1) and the combination (day 8) in cycle 1. Whole exome and transcriptome sequencing was performed using both normal and tumor tissue. Circulating tumor cells (CTC) were evaluated using the CellSearch platform. Results: 24 TNBC pts were enrolled, with 20 pts treated and evaluable for response (8 germline BRCA-mutation positive, 10 non-BRCA mutated, 2 suspected deleterious). Median age was 51 (range 31-63). Median number of prior treatments was 4 (range 1-7). Most frequent drug-related toxicities included: leukopenia (60%), neutropenia (60%), nausea (55%), diarrhea (40%), fatigue (40%), anemia (30%), and vomiting (30%). Best responses were as follows: Germline BRCA-mutant positive 7/8 PR (88%; median number of days on study = 330; range 148-594 days), 1/8 PD (12%); suspected deleterious 2/2 PD (100%); non-BRCA mutated 7/10 SD (70%; median number of days on study = 70; range 42-98 days), 3/10 PD (30%). Exploratory molecular profiling has been performed in a subset of these pts and the results will be presented. EpCAM+ CTC numbers were evaluable in 11 of 22 enrolled pts, and nuclear γH2Ax+, a pharmacodynamic biomarker of DNA damage, was identified in a fraction of CTCs from all 11 of these pts. Conclusions: Veliparib in combination with irinotecan was safe and tolerable in TNBC pts. Although the cohort in this trial is small, the preliminary response rate of 88% in pts with germline BRCA mutation is encouraging and higher than that historically reported with PARP inhibitor monotherapy in this population. Deep molecular profiling among BRCA mutant carriers will be validated in a larger, independent cohort to define potential biomarkers of response. Support: NCI U01-CA062487, NCI U01-CA062490, Komen KG120001, NCI R21-CA135572, and HHSN261200800001E. Citation Format: Patricia M. LoRusso, Sara M. Tolaney, Shukmei Wong, Ralph E. Parchment, Robert J. Kinders, Lihua Wang, Jessica Aldrich, Alice Chen, Diane Durecki, Scott A. Boerner, Tina Guthrie, Adam Bowditch, Lance K. Heilbrun, Mary Jo Pilat, David Craig, Dongpo Cai, Tracy Bell, John Carpten, Geoffrey Shapiro. Combination of the PARP inhibitor veliparib (ABT888) with irinotecan in patients with triple negative breast cancer: Preliminary activity and signature of response. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr CT325. doi:10.1158/1538-7445.AM2015-CT325

Phase I safety, pharmacokinetic and pharmacodynamic study of the poly (ADP-ribose) polymerase inhibitor veliparib with irinotecan in patients with advanced tumors

Purpose: PARP is essential for recognition and repair of DNA damage. In preclinical models, PARP inhibitors modulate topoisomerase I inhibitor–mediated DNA damage. This phase I study determined the MTD, dose-limiting toxicities (DLT), pharmacokinetics (PK), and pharmacodynamics (PD) of veliparib, an orally bioavailable PARP1/2 inhibitor, in combination with irinotecan. Experimental Design: Patients with advanced solid tumors were treated with 100 mg/m2 irinotecan on days 1 and 8 of a 21-day cycle. Twice-daily oral dosing of veliparib (10–50 mg) occurred on days 3 to 14 (cycle 1) and days −1 to 14 (subsequent cycles) followed by a 6-day rest. PK studies were conducted with both agents alone and in combination. Paired tumor biopsies were obtained after irinotecan alone and veliparib/irinotecan to evaluate PARP1/2 inhibition and explore DNA damage signals (nuclear γ-H2AX and pNBS1). Results: Thirty-five patients were treated. DLTs included fatigue, diarrhea, febrile neutropenia, and neutropenia. The MTD was 100 mg/m2 irinotecan (days 1 and 8) combined with veliparib 40 mg twice daily (days −1–14) on a 21-day cycle. Of 31 response-evaluable patients, there were six (19%) partial responses. Veliparib exhibited linear PK, and there were no apparent PK interactions between veliparib and irinotecan. At all dose levels, veliparib reduced tumor poly(ADP-ribose) (PAR) content in the presence of irinotecan. Several samples showed increases in γ-H2AX and pNBS1 after veliparib/irinotecan compared with irinotecan alone. Conclusions: Veliparib can be safely combined with irinotecan at doses that inhibit PARP catalytic activity. Preliminary antitumor activity justifies further evaluation of the combination. Clin Cancer Res; 22(13); 3227–37. ©2016 AACR.

Abstract CT047: Phase 1 dose-escalation study of the CDK inhibitor dinaciclib in combination with the PARP inhibitor veliparib in patients with advanced solid tumors

Background: Although PARP inhibition is effective against HR repair-deficient cancers, efficacy is limited by HR proficiency, whether present de novo or as a result of acquired resistance, prompting HR disrupting strategies to sensitize tumor cells. Inhibition of CDK1 and CDK12 compromise HR by blocking BRCA1 phosphorylation, affecting recruitment to sites of DNA damage, and by reducing HR gene expression, respectively. Dinaciclib is a pan-CDK inhibitor that inhibits both CDK1 and CDK12 at nanomolar potency. We conducted a Phase 1 study combining dinaciclib and veliparib in patients with advanced solid tumors who are not germline BRCA carriers. Methods: A 3+3 design was utilized. Veliparib was administered twice daily continuously in 28-day cycles. Dinaciclib was administered intravenously on days 8 and 22. In part 1A, escalation followed a two-dimensional schema, utilizing doses of dinaciclib between 10 - 45 mg/m2 and veliparib between 20 - 120 mg. In part 1B, veliparib was escalated between 200 mg - 400 mg with dinaciclib maintained at 30 mg/m2. PK and PD assessments were performed at baseline, after veliparib, and after the combination. Preliminary Results: Sixty-three heavily pretreated patients were enrolled in part 1A (n = 39) and 1B (n = 24). Thirty-four patients had breast or gynecologic malignancies. The MTD was 400 mg twice-daily veliparib with dinaciclib at 30 mg/m2. DLTs included G4 neutropenia > 7 days (n =1), febrile neutropenia (n = 1), mucositis (n = 1) and fatigue (n = 1). Common drug-related toxicities were neutropenia (78%), nausea (75%), fatigue (67%), electrolyte abnormalities (59%), elevated liver function tests (57%), diarrhea (52%), lymphopenia (52%), anemia (43%), dehydration (37%), anorexia (30%), vomiting (29%), hypoalbuminemia (29%), dizziness (29%), headache (22%), mucositis (18%), elevated creatinine (16%), alopecia (16%), thrombocytopenia (14%), abdominal pain (13%), insomnia (13%), and dysgeusia (11%). The median number of cycles completed was 2 (r: 1 - 10). One patient with TNBC achieved complete resolution of axillary adenopathy lasting > 8 months. Twenty-four patients (38%) had stable disease as the best response, with 9 progression-free > 4 months (TNBC, gynecologic and thymic ca). Paired tumor biopsies from one patient demonstrated reduced Ki-67 and increased gamma-H2AX staining after combination treatment compared to after veliparib alone. Conclusions: Dinaciclib administered at doses known to produce PD effects is tolerable with full dose veliparib. Anti-tumor activity is limited in non-BRCA carriers, possibly related to intermittent administration of a CDK inhibitor with known short half-life. Additional patients are being enrolled utilizing dinaciclib in more dose-intense schedules. Citation Format: Geoffrey I. Shapiro, Khanh T. Do, Sara M. Tolaney, John F. Hilton, James M. Cleary, Andrew Wolanski, Brian Beardslee, Faith Hassinger, Ketki Bhushan, Dongpo Cai, Elizabeth Downey, Solida Pruitt-Thompson, Suzanne M. Barry, Bose Kochupurakkal, Joseph Geradts, Christine Unitt, Alan D. D9Andrea, Alona Muzikansky, Richard Piekarz, L. Austin Doyle, Jeffrey Supko. Phase 1 dose-escalation study of the CDK inhibitor dinaciclib in combination with the PARP inhibitor veliparib in patients with advanced solid tumors [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 CT047. doi:10.1158/1538-7445.AM2017-CT047

Phase I safety, pharmacokinetic and pharmacodynamic study of the poly (ADP-ribose) polymerase inhibitor veliparib with irinotecan in patients with advanced solid tumors

Purpose: PARP is essential for recognition and repair of DNA damage. In preclinical models, PARP inhibitors modulate topoisomerase I inhibitor–mediated DNA damage. This phase I study determined the MTD, dose-limiting toxicities (DLT), pharmacokinetics (PK), and pharmacodynamics (PD) of veliparib, an orally bioavailable PARP1/2 inhibitor, in combination with irinotecan. Experimental Design: Patients with advanced solid tumors were treated with 100 mg/m2 irinotecan on days 1 and 8 of a 21-day cycle. Twice-daily oral dosing of veliparib (10–50 mg) occurred on days 3 to 14 (cycle 1) and days −1 to 14 (subsequent cycles) followed by a 6-day rest. PK studies were conducted with both agents alone and in combination. Paired tumor biopsies were obtained after irinotecan alone and veliparib/irinotecan to evaluate PARP1/2 inhibition and explore DNA damage signals (nuclear γ-H2AX and pNBS1). Results: Thirty-five patients were treated. DLTs included fatigue, diarrhea, febrile neutropenia, and neutropenia. The MTD was 100 mg/m2 irinotecan (days 1 and 8) combined with veliparib 40 mg twice daily (days −1–14) on a 21-day cycle. Of 31 response-evaluable patients, there were six (19%) partial responses. Veliparib exhibited linear PK, and there were no apparent PK interactions between veliparib and irinotecan. At all dose levels, veliparib reduced tumor poly(ADP-ribose) (PAR) content in the presence of irinotecan. Several samples showed increases in γ-H2AX and pNBS1 after veliparib/irinotecan compared with irinotecan alone. Conclusions: Veliparib can be safely combined with irinotecan at doses that inhibit PARP catalytic activity. Preliminary antitumor activity justifies further evaluation of the combination. Clin Cancer Res; 22(13); 3227–37. ©2016 AACR.