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Supplementary figure legends and supplementary materials and methods

Table S1. Summary of CC-90003 specificity data resulting from cellular and biochemical assays. Table S2. Response of BRAF mutant cell lines to CC-90003 ERK 1/2 inhibition. Table S3. Summary of dose finding efficacy study in HCT-116 xenograft model. Figure S1. CC-90003 Mass spectrometry analysis of CC-90003 covalent ERK1/2 binding. Figure S2. CC-90003 Inhibits proliferation and induces apoptosis in a panel of KRAS mutant Lung, Pancreatic and Colorectal cancer cell lines. Figure S3. Assessment of ERK inhibitor PD markers in BRAF and KRAS mutant Cancer Models. Figure S4. CC-90003 inhibits proliferation of a subset of BRAF and KRAS mutant cell lung cancer lines and induces cell death more robustly, compared to published inhibitors. Figure S5. CC-90003 inhibits colony formation in a set of KRAS mutant PDX models ex vivo. Figure S6. CC-90003 does not reduce body weights of animals bearing KRAS mutant PDX derived tumors. Figure S7. Genes differentially expressed in CXF-243 PDX model compared to other models used in the study. Figure S8. Genes differentially expressed in CXF-243, CXF-975 and CXF-1034 PDX models compared to other models used in the study. Figure S9. Combination of CC-90003 with EGFR inhibitor Afatinib and NK inhibitor JNK-IN-8. Figure S10. Quantification of Western Blot data in Figure 4. Figure S11. CC-90003 does not reduce body weights of animals bearing LXFA-1674 KRAS mutant lung cancer PDX derived tumors. Figure S12. Regrowth of LXFA-1674 KRAS mutant lung cancer PDX derived tumors following discontinuation of treatment by Docetaxel and CC-90003 combination with Docetaxel. Figure S13. CC-90003 alone and in combination with Docetaxel modulates stemness program in ex vivo serial passaging Mammocult assay.

As a critical signaling node, ERK1/2 are attractive drug targets, particularly in tumors driven by activation of the MAPK pathway. Utility of targeting the MAPK pathway has been demonstrated by clinical responses to inhibitors of MEK1/2 or RAF kinases in some mutant BRAF-activated malignancies. Unlike tumors with mutations in BRAF, those with mutations in KRAS (>30% of all cancers and >90% of certain cancer types) are generally not responsive to inhibitors of MEK1/2 or RAF. Here, a covalent ERK1/2 inhibitor, CC-90003, was characterized and shown to be active in preclinical models of KRAS-mutant tumors. A unique occupancy assay was used to understand the mechanism of resistance in a KRAS-mutant patient-derived xenograft (PDX) model of colorectal cancer. Finally, combination of CC-90003 with docetaxel achieved full tumor regression and prevented tumor regrowth after cessation of treatment in a PDX model of lung cancer. This effect corresponded to changes in a stemness gene network, revealing a potential effect on tumor stem cell reprograming.Implications:Here, a covalent ERK1/2 inhibitor (CC-90003) is demonstrated to have preclinical efficacy in models of KRAS-mutant tumors, which present a therapeutic challenge for currently available therapies.

PDF file - 30K, Estimated maximal concentration (Cmax) and total area under the concentration-time curve (AUClast) of total and free oxaliplatin with and without vismodegib

PDF file - 25K, A comparison of steady-state 5-FU concentrations with and without vismodegib co-treatment

Correlation analysis between pharmacodynamic and pharmacokinetic findings in part 1

Mean paclitaxel plasma concentrations following nab-paclitaxel administration alone or with CC-486 in part 1 arm B

Table S1. CC-486 Plus Carboplatin Dose Levels in Part 1 Arm A Table S2. CC-486 Plus nab-Paclitaxel Dose Levels in Part 1 Arm B Table S3. CC-486 Dose Levels in Part 1 Arm C Table S4. Number of Patients With Treatment Emergent Adverse Events for Part 1 Table S5. Number of Patients With Treatment-Emergent Adverse Events in Any Treatment Arm in Part 2 Table S6. Mean CC-486 Plasma Pharmacokinetic Parameters Table S7. Efficacy of CC-486 Alone or in Combination With Carboplatin or nab-Paclitaxel in Part 2 Table S8. Prior Treatment History of NPV Patients

Mean total carboplatin plasma concentration following carboplatin administration alone or with CC-486 in part 1 arm A

PDF file - 33K, Estimated maximal concentration (Cmax) and total area under the concentration-time curve (AUClast) of irinotecan (and metabolites SN-38 and SN-38G) with and without vismodegib

PDF file - 25K, A comparison of serum concentrations of bevacizumab with and without vismodegib co-treatment

PDF file - 57K, A summary of primer/probes used for qRT-PCR profiling of Hh pathway genes

Purpose: Vismodegib, a Hedgehog pathway inhibitor, has preclinical activity in colorectal cancer (CRC) models. This trial assessed the efficacy, safety, and pharmacokinetics of adding vismodegib to first-line treatment for metastatic CRC (mCRC).Experimental design: Patients were randomized to receive vismodegib (150 mg/day orally) or placebo, in combination with FOLFOX or FOLFIRI chemotherapy plus bevacizumab (5 mg/kg) every 2 weeks until disease progression or intolerable toxicity. The primary endpoint was progression-free survival (PFS). Key secondary objectives included evaluation of predictive biomarkers and pharmacokinetic drug interactions.Results: A total of 199 patients with mCRC were treated on protocol (124 FOLFOX, 75 FOLFIRI). The median PFS hazard ratio (HR) for vismodegib treatment compared with placebo was 1.25 (90% CI: 0.89–1.76; P = 0.28). The overall response rates for placebo-treated and vismodegib-treated patients were 51% (90% CI: 43–60) and 46% (90% CI: 37–55), respectively. No vismodegib-associated benefit was observed in combination with either FOLFOX or FOLFIRI. Increased tumor tissue Hedgehog expression did not predict clinical benefit. Grade 3 to 5 adverse events reported for more than 5% of patients that occurred more frequently in the vismodegib-treated group were fatigue, nausea, asthenia, mucositis, peripheral sensory neuropathy, weight loss, decreased appetite, and dehydration. Vismodegib did not alter the pharmacokinetics of FOLFOX, FOLFIRI, or bevacizumab.Conclusions: Vismodegib does not add to the efficacy of standard therapy for mCRC. Compared with placebo, treatment intensity was lower for all regimen components in vismodegib-treated patients, suggesting that combined toxicity may have contributed to lack of efficacy. Clin Cancer Res; 19(1); 258–67. ©2012 AACR.

Pharmacodynamic Assessments

PDF file - 24K, A comparison of vismodegib steady-state plasma concentrations (Css) between patients receiving FOLFOX and FOLFIRI

PDF file - 61K

Purpose: This large two-part, three-arm phase I study examined the safety and tolerability of CC-486 (an oral formulation of azacitidine, a hypomethylating agent) alone or in combination with the cytotoxic agents, carboplatin or nab-paclitaxel, in patients with advanced unresectable solid tumors.Patients and Methods: Part 1 (n = 57) was a dose escalation of CC-486 alone (arm C) or with carboplatin (arm A) or nab-paclitaxel (arm B). The primary endpoint was safety, MTD, and recommended part 2 dose (RP2D) of CC-486. In part 2 (n = 112), the primary endpoint was the safety and tolerability of CC-486 administered at the RP2D for each treatment arm, in tumor-specific expansion cohorts. Secondary endpoints included pharmacokinetics, pharmacodynamics, and antitumor activity of CC-486.Results: At pharmacologically active doses CC-486 in combination with carboplatin or nab-paclitaxel had a tolerable safety profile and no drug–drug interactions. The CC-486 RP2D was determined as 300 mg (every day, days 1–14/21) in combination with carboplatin (arm A) or as monotherapy (arm C); and 200 mg in the same dosing regimen in combination with nab-paclitaxel (arm B). Albeit limited by the small sample size, CC-486 monotherapy resulted in partial responses (three/eight) and stable disease (four/eight) in patients with nasopharyngeal cancer. Three of the stable disease responses lasted more than 150 days.Conclusions: CC-486 is well tolerated alone or in combination with carboplatin or nab-paclitaxel. Exploratory analyses suggest clinical activity of CC-486 monotherapy in nasopharyngeal cancer and provided the basis for an ongoing phase II clinical trial (ClinicalTrials.gov identifier: NCT02269943). Clin Cancer Res; 24(17); 4072–80. ©2018 AACR.

As a critical signaling node, ERK1/2 are attractive drug targets, particularly in tumors driven by activation of the MAPK pathway. Utility of targeting the MAPK pathway has been demonstrated by clinical responses to inhibitors of MEK1/2 or RAF kinases in some mutant BRAF-activated malignancies. Unlike tumors with mutations in BRAF, those with mutations in KRAS (>30% of all cancers and >90% of certain cancer types) are generally not responsive to inhibitors of MEK1/2 or RAF. Here, a covalent ERK1/2 inhibitor, CC-90003, was characterized and shown to be active in preclinical models of KRAS-mutant tumors. A unique occupancy assay was used to understand the mechanism of resistance in a KRAS-mutant patient-derived xenograft (PDX) model of colorectal cancer. Finally, combination of CC-90003 with docetaxel achieved full tumor regression and prevented tumor regrowth after cessation of treatment in a PDX model of lung cancer. This effect corresponded to changes in a stemness gene network, revealing a potential effect on tumor stem cell reprograming. Implications: Here, a covalent ERK1/2 inhibitor (CC-90003) is demonstrated to have preclinical efficacy in models of KRAS-mutant tumors, which present a therapeutic challenge for currently available therapies.

Background: Spleen tyrosine kinase (SYK) is a component of both lymphoid and myeloid cell signaling pathways and has been implicated in the pathogenesis of a subset of acute myeloid leukemia (AML) defined by dysregulated expression of the HOXA9 and MEIS1 transcription factors. Entospletinib (ENTO) is an oral, selective SYK inhibitor that is acceptably tolerated when administered with intensive induction and consolidation in newly diagnosed AML patients. In a phase 2 study, following induction with cytarabine and daunorubicin (7+3) plus ENTO, higher rates of complete response (CR) or CR with incomplete hematologic recovery (CRi) were observed in patients with rearrangements of the KMT2A (MLL) gene (MLL-r) and mutations of the nucleophosmin 1 (NPM1) gene, both of which are associated with aberrant expression of HOXA9 and MEIS1, as compared to patients without these mutations. In an exploratory analysis, patients with HOXA9/MEIS1 expression levels above the median experienced superior overall survival (OS) as compared to patients with expression levels below the median. In the AGILITY trial, we hypothesize that the addition of ENTO to intensive induction/consolidation in newly diagnosed patients with NPM1-mutated AML will improve the rate of CR without evidence of measurable residual disease (MRD-negative CR) post-induction and duration of event-free survival (EFS). Methods: AGILITY will be a global, multi-center, double-blind, placebo-controlled trial of ENTO in combination with cytarabine plus daunorubicin or idarubicin induction (7+3) and age-adjusted high-dose cytarabine (HiDAC) consolidation in newly diagnosed AML patients aged 18-75 years who are candidates for intensive induction and harbor a documented NPM1 mutation based on local or central mutation testing. Patients with co-mutated FLT3 (internal tandem duplication or tyrosine kinase domain) and for whom midostaurin with 7+3 is indicated are excluded. Patients will be stratified based on age ( An independent data-monitoring committee will monitor emerging safety and efficacy data from this trial on an ongoing basis. Disclosures Byrd: Vincerx Pharmaceuticals: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Novartis, Trillium, Astellas, AstraZeneca, Pharmacyclics, Syndax: Consultancy, Honoraria; Newave: Membership on an entity's Board of Directors or advisory committees. Cortes: Bristol Myers Squibb, Daiichi Sankyo, Jazz Pharmaceuticals, Astellas, Novartis, Pfizer, Takeda, BioPath Holdings, Incyte: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Bio-Path Holdings, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Sun Pharma: Consultancy, Research Funding. Minden: Astellas: Consultancy. Oellerich: Roche: Consultancy; Gilead: Research Funding; Kronos Bio, Inc.: Consultancy; Merck KGaA: Consultancy, Research Funding. Stein: Syros Pharmaceuticals, Inc.: Consultancy; Daiichi Sankyo: Consultancy; PinotBio: Consultancy; Celgene: Consultancy; Bristol Myers Squibb: Consultancy; Jazz Pharmaceuticals: Consultancy; Foghorn Therapeutics: Consultancy; Blueprint Medicines: Consultancy; Gilead Sciences, Inc.: Consultancy; Abbvie: Consultancy; Janssen Pharmaceuticals: Consultancy; Genentech: Consultancy; Syndax Pharmaceuticals: Consultancy; Agios Pharmaceuticals, Inc: Consultancy; Novartis: Consultancy; Astellas: Consultancy. Elder: PharPoint Research, Inc.: Current Employment. Kumar: Kronos Bio, Inc.: Current Employment, Current equity holder in publicly-traded company. Bray: Kronos Bio, Inc.: Consultancy. DiMartino: Kronos Bio, Inc.: Current Employment, Current equity holder in publicly-traded company. Stock: Pfizer: Consultancy, Honoraria, Research Funding; amgen: Honoraria; agios: Honoraria; jazz: Honoraria; kura: Honoraria; kite: Honoraria; morphosys: Honoraria; servier: Honoraria; syndax: Consultancy, Honoraria; Pluristeem: Consultancy, Honoraria. OffLabel Disclosure: Entospletinib is an investigational therapy

Purpose: This large two-part, three-arm phase I study examined the safety and tolerability of CC-486 (an oral formulation of azacitidine, a hypomethylating agent) alone or in combination with the cytotoxic agents, carboplatin or nab-paclitaxel, in patients with advanced unresectable solid tumors. Patients and Methods: Part 1 (n = 57) was a dose escalation of CC-486 alone (arm C) or with carboplatin (arm A) or nab-paclitaxel (arm B). The primary endpoint was safety, MTD, and recommended part 2 dose (RP2D) of CC-486. In part 2 (n = 112), the primary endpoint was the safety and tolerability of CC-486 administered at the RP2D for each treatment arm, in tumor-specific expansion cohorts. Secondary endpoints included pharmacokinetics, pharmacodynamics, and antitumor activity of CC-486. Results: At pharmacologically active doses CC-486 in combination with carboplatin or nab-paclitaxel had a tolerable safety profile and no drug–drug interactions. The CC-486 RP2D was determined as 300 mg (every day, days 1–14/21) in combination with carboplatin (arm A) or as monotherapy (arm C); and 200 mg in the same dosing regimen in combination with nab-paclitaxel (arm B). Albeit limited by the small sample size, CC-486 monotherapy resulted in partial responses (three/eight) and stable disease (four/eight) in patients with nasopharyngeal cancer. Three of the stable disease responses lasted more than 150 days. Conclusions: CC-486 is well tolerated alone or in combination with carboplatin or nab-paclitaxel. Exploratory analyses suggest clinical activity of CC-486 monotherapy in nasopharyngeal cancer and provided the basis for an ongoing phase II clinical trial (ClinicalTrials.gov identifier: NCT02269943). Clin Cancer Res; 24(17); 4072–80. ©2018 AACR.

Purpose: PRO95780 is a fully human IgG1 monoclonal antibody that triggers the extrinsic apoptosis pathway through death receptor 5. This first-in-human study assessed the safety, tolerability, pharmacokinetics, and any early evidence of efficacy of PRO95780 in patients with advanced malignancies. Experimental Design: Target concentrations were predicted to occur at 10 mg/kg. Patients received up to eight cycles of PRO95780 i.v. using a 3+3 dose escalation design at 1 to 20 mg/kg every 14 days (every 28 days in cycle 1; stage 1), with cohort expansion at either the maximum tolerated dose or 10 mg/kg, whichever was lower (stage 2). Patients were evaluated for response every other cycle. Results: The maximum tolerated dose was not reached within this study. Four (8%) of 50 patients reported adverse events of greater than grade 2 at least possibly related to PRO95780, including 2 patients with reversible grade 3 transaminase elevation. The mean terminal half-life was 8.8 to 19.3 days, with dose-dependent increases in exposure (peak plasma concentration and area under the concentration) across 1 to 15 mg/kg. Most patients treated with 10 mg/kg or above achieved trough concentration above the target efficacious concentration at day 15 with moderate accumulation after multiple doses. No objective responses occurred, although three minor responses were observed in patients with colorectal and granulosa cell ovarian cancers (each treated with 4 mg/kg) and chondrosarcoma (10 mg/kg). Conclusions: PRO95780 is safe and well tolerated at doses up to 20 mg/kg. Evidence of activity was noted in several different tumor types at 4 and 10 mg/kg. Pharmacokinetic analysis supports a dosing regimen of 10 to 15 mg/kg every 2 to 3 weeks. Clin Cancer Res; 16(4); 1256–63

To evaluate the effects of human immunodeficiency virus type 1 (HIV-1) infection on the loss of factor VIII alloantibodies, we identified 77 patients with a history of inhibitors from among a large cohort of HIV-1-infected participants enrolled in a natural history study of HIV-1 infection in hemophilia. Fifty-six patients were high responders with inhibitors titers greater than 5 Bethesda Units (BU) measured on at least one occasion. From May 1985 to December 1989, 13 of the high-responder patients were rechallenged with factor VIII concentrates after several years of treatment with other plasma products. All exhibited excellent hemostasis upon reinstitution of factor VIII. Seven of the 13 patients (11.3–46.3 years of age) were in the advanced stages of HIV-1 infection at the time of rechallenge. Inhibitor titers measured subsequent to the reinstitution of factor VIII were consistently less than 1 BU in five of these seven patients. The remaining six patients (6.1–57.5 years of age) had mild to moderate CD4+ lymphocyte depletion (absolute CD4+ cells: 262–935/ mm3) at the time of factor VIII rechallenge. Follow-up inhibitor titers were negative 7–42 months after consistent factor VIII use in these six patients. The lack of anamnestic response to factor VIII in all 13 patients who were rechallenged indicates that HIV-1-infected patients who have a history of high-responder inhibitors frequently benefit from the reintroduction of factor VIII use for the control of bleeding, regardless of their stage of HIV-1 disease. © 1993 Wiley-Liss, Inc.

LOSS OF NATURALLY ACQUIRED HEPATITIS B SURFACE ANTI-BODY (ANTI-HBs) IN TWO HEMOPHILIACS WITH AIDS RELATED COMPLEX