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Redacted protocol for supplementary files

Supplementary Figure 4 shows pharmacokinetic parameters for guadecitabine and decitabine in the dose escalation phase

Purpose:Preclinical data indicate that DNA methyltransferase inhibition will circumvent cisplatin resistance in various cancers.Patient and Methods:SPIRE comprised a dose-escalation phase for incurable metastatic solid cancers, followed by a randomized dose expansion phase for neoadjuvant treatment of T2–4a N0 M0 bladder urothelial carcinoma. The primary objective was a recommended phase II dose (RP2D) for guadecitabine combined with gemcitabine and cisplatin. Treatment comprised 21-day gemcitabine and cisplatin cycles (cisplatin 70 mg/m2, i.v., day 8 and gemcitabine 1,000 mg/m2, i.v., days 8 + 15). Guadecitabine was injected subcutaneously on days 1–5, within escalation phase cohorts, and to half of 20 patients in the expansion phase. Registration ID: ISRCTN 16332228.Results:Within the escalation phase, dose-limiting toxicities related predominantly to myelosuppression requiring G-CSF prophylaxis from cohort 2 (guadecitabine 20 mg/m2, days 1–5). The most common grade ≥3 adverse events in 17 patients in the dose-escalation phase were neutropenia (76.5%), thrombocytopenia (64.7%), leukopenia (29.4%), and anemia (29.4%). Addition of guadecitabine to gemcitabine and cisplatin in the expansion phase resulted in similar rates of severe hematologic adverse events, similar cisplatin dose intensity, but modestly reduced gemcitabine dose intensity. Radical treatment options after chemotherapy were not compromised. Pharmacodynamics evaluations indicated guadecitabine maximal target effect at the point of cisplatin administration. Pharmacokinetics were consistent with prior data. No treatment-related deaths occurred.Conclusions:The guadecitabine RP2D was 20 mg/m2, days 1–5, in combination with gemcitabine and cisplatin and required GCSF prophylaxis. Gene promoter methylation pharmacodynamics are optimal with this schedule. Addition of guadecitabine to gemcitabine and cisplatin was tolerable, despite some additional myelosuppression, and warrants further investigation to assess efficacy.

Supplementary Tables and Methods

Supplementary Figure 1 shows a schematic of the treatment cycle

Supplementary Figure 2 shows CONSORT diagrams for both phases of the trial

Supplementary Figure 3 shows promotor methylation status for the indicated genes in cfDNA

Melphalan is an alkylating agent used as part of conditioning prior to pediatric hematopoietic cell transplantation (HCT). We performed a single-center, prospective pharmacokinetic study of 37 pediatric patients undergoing HCT from March 2015 to 2019. The primary objective was to develop and validate a population pharmacokinetic model for melphalan in a diverse group of pediatric HCT recipients. Nonlinear mixed-effects modeling was implemented to describe plasma concentration-time data of melphalan. A 2-compartment, proportional error model with weight on clearance best fit the data. Final parameter estimates were clearance, 19.1 L/h/25 kg; volume of the central compartment, 8.5 L/25 kg; volume of the peripheral compartment, 5.8 L/25 kg; and intercompartmental clearance 12.4 L/h/25 kg. Residual unexplained variability was low, at 12.5%. Results suggest the empiric weight-based dosing (mg/kg) used in children12 kg or 2 years of age may result in subtherapeutic exposure. Model-based dosing of melphalan in pediatric HCT may help inform individualized dosing strategies to improve clinical outcomes and limit drug-related adverse events in pediatric HCT recipients.

9085 Background: Aberrant activation of the RAS-RAF-MEK-ERK pathway is common in human cancers. This is an open-label Phase 1 study of ASTX029, a dual-mechanism extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor, in subjects with relapsed/refractory solid tumors (NCT03520075). Methods: The primary objective is to identify a recommended Phase 2 dose. Subjects with relapsed/refractory solid tumors were eligible for Phase 1A with any molecular feature and for Phase 1B if the tumor demonstrated RAS or BRAF mutations. ASTX029 was administered orally daily on a continuous basis in 21-day cycles. Phase 1A was a modified 3+3 dose-escalation design based on dose-limiting toxicity (DLT) events. Phase 1B subjects were treated at the recommended dose for expansion (RDE) based on emerging safety, pharmacokinetic (PK), and pharmacodynamic (PD) data. Disease response was evaluated by RECIST v1.1. Results: 76 subjects were treated with at least one dose of ASTX029 in Phase 1A (n = 56) and Phase 1B (n = 20). In Phase 1A, ASTX029 was evaluated from 10 mg to 280 mg daily. Two subjects experienced grade 2 central serous retinopathy (CSR) within a few days of dosing at the 280 mg daily dose level (one event was declared a DLT). Both subjects recovered to baseline within days of dose interruption. CSR is an expected AE based on the class of drugs. At the selected RDE dose level of 200 mg daily, the mean PK exposure was 109% of target exposure (13,022 ng*hr/ml), defined as the level expected to have biological activity based on mouse models. As of the data cut-off of February 7, 2022, the most frequent grade ≥2 AEs experienced by subjects (≥5%) assessed as related to ASTX029 included ocular AEs (n = 6: all Grade 2); nausea (n = 7: all Grade 2); diarrhea (n = 6: 5 Grade 2, 1 Grade 3); fatigue (n = 4: all Grade 2); rash (n = 4, 3 Grade 2, 1 Grade 3). There were 52 serious AEs, all unrelated to ASTX029 except for one subject with Grade 3 malaise. Four subjects had a partial response, including KRAS-G12A BRAF-D549N non-small cell lung cancer (NSCLC; Phase 1A: 120 mg treated 20.0 months); KRAS-G12D pancreatic cancer (Phase 1A: 200 mg treated 2.1 months); KRAS-G13D NSCLC (Phase 1B; treated 10.6 months); KRAS-G12S NSCLC (Phase 1B; treated 10.4 months and ongoing). In all, two partial responses were observed out of 3 NSCLC subjects enrolled in Phase 1B. Phospho-ERK and phospho-RSK were evaluated for PD effect on fresh tumor biopsies obtained at baseline and cycle 2. A PD effect and decreased cell proliferation (Ki-67) were observed in 6 of 9 and 3 of 8 evaluable Phase 1B samples, respectively. The most common reason for ASTX029 discontinuation was disease progression. Conclusions: This Phase 1 study of the ERK1/2 inhibitor ASTX029 has identified a dose level of 200 mg daily continuously for investigation in the Phase 2 study. PK and PD data suggest target exposures are achieved with preliminary clinical activity, especially in KRAS-mutated NSCLC. Clinical trial information: NCT03520075.

Purpose: Preclinical data indicate that DNA methyltransferase inhibition will circumvent cisplatin resistance in various cancers. Patient and Methods: SPIRE comprised a dose-escalation phase for incurable metastatic solid cancers, followed by a randomized dose expansion phase for neoadjuvant treatment of T2–4a N0 M0 bladder urothelial carcinoma. The primary objective was a recommended phase II dose (RP2D) for guadecitabine combined with gemcitabine and cisplatin. Treatment comprised 21-day gemcitabine and cisplatin cycles (cisplatin 70 mg/m2, i.v., day 8 and gemcitabine 1,000 mg/m2, i.v., days 8 + 15). Guadecitabine was injected subcutaneously on days 1–5, within escalation phase cohorts, and to half of 20 patients in the expansion phase. Registration ID: ISRCTN 16332228. Results: Within the escalation phase, dose-limiting toxicities related predominantly to myelosuppression requiring G-CSF prophylaxis from cohort 2 (guadecitabine 20 mg/m2, days 1–5). The most common grade ≥3 adverse events in 17 patients in the dose-escalation phase were neutropenia (76.5%), thrombocytopenia (64.7%), leukopenia (29.4%), and anemia (29.4%). Addition of guadecitabine to gemcitabine and cisplatin in the expansion phase resulted in similar rates of severe hematologic adverse events, similar cisplatin dose intensity, but modestly reduced gemcitabine dose intensity. Radical treatment options after chemotherapy were not compromised. Pharmacodynamics evaluations indicated guadecitabine maximal target effect at the point of cisplatin administration. Pharmacokinetics were consistent with prior data. No treatment-related deaths occurred. Conclusions: The guadecitabine RP2D was 20 mg/m2, days 1–5, in combination with gemcitabine and cisplatin and required GCSF prophylaxis. Gene promoter methylation pharmacodynamics are optimal with this schedule. Addition of guadecitabine to gemcitabine and cisplatin was tolerable, despite some additional myelosuppression, and warrants further investigation to assess efficacy.

Introduction: The combination of a DNA methyltransferase inhibitor (DNMTi; parenteral azacitidine or decitabine) with the BCL2 inhibitor venetoclax is a newly established standard-of-care regimen for patients with newly diagnosed acute myeloid leukemia (AML) ineligible to receive intensive induction chemotherapy (DiNardo et al, 2020). Replacing the parenteral DNMTi with an oral DNMTi with equivalent exposure may provide the benefit of reducing patient and caregiver burden of chronic parenteral therapy, and may help responding patients stay on treatment longer. ASTX727 (a fixed-dose combination of decitabine 35 mg and cedazuridine 100 mg) is an oral DNMTi that provides equivalent exposure to its parenteral DNMTi at standard dosing (Savona et al, 2020) and is under evaluation in combination with venetoclax as an all-oral regimen. Methods: This is an ongoing Phase 1 study being conducted at 7 US medical centers (ClinicalTrials.gov NCT04657081). Newly diagnosed AML patients 75 years or older, or with comorbidities that preclude use of intensive induction chemotherapy are eligible. The primary objective is to evaluate the effect of ASTX727 on the PK of venetoclax. Key secondary objectives are to evaluate the effect of venetoclax on the PK of ASTX727, and to determine the safety and efficacy profile for the combination. For Cycle 2 and beyond, ASTX727 is administered orally daily on days 1-5 and venetoclax 400 mg is administered orally daily on days 1-28 of 28-day cycles. For Cycle 1, ASTX727 is given in the same dose schedule while venetoclax is given as a ramp-up on days 1 and 2 according to the venetoclax US prescribing information (USPI); therefore, the PK studies are conducted during Cycle 2. Delay of subsequent cycles and venetoclax dose modifications for hematologic toxicities and anti-fungal concomitant medications follow the venetoclax USPI. Response assessments are evaluated using the 2017 ELN criteria (Döhner et al, 2017) and the CRh criterion (complete response [CR] with partial hematologic recovery) defined as those patients achieving marrow CR criteria but not peripheral blood count criteria and demonstrating an absolute neutrophil count >500/μL and platelet >50,000/μL (Kantarjian et al, 2017). Results: At the data cut-off date of August 1, 2021, 15 AML patients have enrolled and received study treatment. Median age is 78 years (range 66 - 84) and 9 (60%) are males. Of the 12 patients with data, 2 (17%), 6 (50%), and 4 (33%) patients are in the favorable, intermediate, and adverse risk ELN categories, respectively. Of the 15 dosed patients, 6 (40%) are diagnosed with AML with myelodysplasia-related changes. The median duration of exposure is 2 cycles (range 1-5) and 1.7 months (range 0.8-5.6). The most common adverse events (AEs) of Grade 2 or higher occurring in ≥10% of patients include neutropenia (5, 33%), febrile neutropenia (2, 13%), anemia (2, 13%), thrombocytopenia (2, 13%), vomiting (2, 13%), pneumonia (2, 13%), peripheral edema (2, 13%), hypertension (2, 13%) and vascular access complication (2, 13%). There are 17 serious AEs experienced in 7 patients; a grade 3 pneumonia and a grade 3 dysphagia are the only serious AEs assessed as related to ASTX727 and/or venetoclax and both events occurred in the same patient. Both AEs were part of the patient's medical history. Two deaths have occurred to date: one patient due to rapidly progressive disease during Cycle 2 and one patient who achieved a best response of CRh transitioned to hospice due to progressive multiple myeloma. Of 9 patients with response assessments and evaluable data, 3 (33%) achieved CR and 4 (44%) achieved CRh as the best response for a composite CR+CRh rate of 78%. Preliminary PK data available from 9 patients show venetoclax exposures are not affected by coadministration of ASTX727and are similar to historical data. Exposures of decitabine and cedazuridine are consistent with the range seen in previous studies. Updated PK, safety, and efficacy data will be provided in December 2021. Conclusions: A preliminary analysis of ASTX727 and venetoclax combination therapy in patients with newly diagnosed AML unfit for intensive induction chemotherapy demonstrate expected PK data, and a similar safety and efficacy profile to the approved combination therapy of a DNMTi and venetoclax. These preliminary data suggest that an all-oral regimen of a DNMTi in combination with venetoclax is feasible and should be investigated further. Disclosures Mannis: AbbVie, Agios, Astellas Pharma, Bristol Myers Squibb, Genentech, MacroGenics, Pfizer, and Stemline: Consultancy; Astex, Forty Seven Inc/Gilead, Glycomimetics, and Jazz Pharmaceuticals: Research Funding. Griffiths: Novartis: Honoraria; Apellis Pharmaceuticals: Research Funding; Alexion Pharmaceuticals: Consultancy, Research Funding; Boston Biomedical: Consultancy; Genentech: Research Funding; Abbvie: Consultancy, Honoraria; Takeda Oncology: Consultancy, Honoraria; Astex Pharmaceuticals: Honoraria, Research Funding; Taiho Oncology: Consultancy, Honoraria; Celgene/Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding. Savona: Geron: Consultancy, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; CTI: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS-Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; NOVARTIS: Consultancy, Membership on an entity's Board of Directors or advisory committees; Ryvu: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sierra Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Taiho: Consultancy, Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; ALX Oncology: Research Funding; Astex: Research Funding; Incyte: Research Funding. Odenike: Celgene, Incyte, AstraZeneca, Astex, NS Pharma, AbbVie, Gilead, Janssen, Oncotherapy, Agios, CTI/Baxalta, Aprea: Research Funding; AbbVie, Celgene, Impact Biomedicines, Novartis, Taiho Oncology, Takeda: Consultancy. Roboz: MEI Pharma - IDMC Chair: Consultancy; Amgen: Consultancy; Jazz: Consultancy; Celgene: Consultancy; Agios: Consultancy; Janssen: Consultancy; Astex: Consultancy; Daiichi Sankyo: Consultancy; Janssen: Research Funding; AstraZeneca: Consultancy; Blueprint Medicines: Consultancy; Bayer: Consultancy; Actinium: Consultancy; AbbVie: Consultancy; Jasper Therapeutics: Consultancy; Helsinn: Consultancy; Glaxo SmithKline: Consultancy; Mesoblast: Consultancy; Bristol Myers Squibb: Consultancy; Novartis: Consultancy; Astellas: Consultancy; Otsuka: Consultancy; Pfizer: Consultancy; Roche/Genentech: Consultancy. Dillingham: Astex Pharmaceuticals, Inc.: Current Employment. Wason: Astex Pharmaceuticals, Inc.: Current Employment. Zhu: Astex Pharmaceuticals, Inc.: Current Employment. Chan: Astex Pharmaceuticals, Inc.: Current Employment. Keer: Astex Pharmaceuticals, Inc.: Current Employment. Oganesian: Astex Pharmaceuticals, Inc.: Current Employment. Dao: Astex Pharmaceuticals, Inc.: Current Employment. DiNardo: AbbVie: Consultancy, Research Funding; Agios/Servier: Consultancy, Honoraria, Research Funding; Novartis: Honoraria; Takeda: Honoraria; Notable Labs: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; ImmuneOnc: Honoraria, Research Funding; GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Research Funding; Foghorn: Honoraria, Research Funding; Forma: Honoraria, Research Funding; Celgene, a Bristol Myers Squibb company: Honoraria, Research Funding. OffLabel Disclosure: 1. Inqovi (35 mg decitabine and 100 mg cedazuridine) is a prescription medicine approved in the United States to treat adults with myelodysplastic syndromes and chronic myelomonocytic leukemia. In this study, Inqovi is referred as ASTX727 due to the off-label investigational use in combination with venetoclax in adults with newly diagnosed acute myeloid leukemia 75 years or older, or who have comorbidities that preclude use of intensive induction chemotherapy. 2. Venclexta is a prescription medicine approved in the United States for: 1) adults with chronic lymphocytic leukemia or small lymphocytic lymphoma; 2) adults with newly diagnosed acute myeloid leukemia 75 years or older, or who have comorbidities that preclude use of intensive induction chemotherapy, in combination with azacitidine, decitabine, or low-dose cytarabine. In this study, Venclexta is referred as venetoclax due to the off-label investigational use in combination with ASTX727 in adults with newly diagnosed acute myeloid leukemia 75 years or older, or who have comorbidities that preclude use of intensive induction chemotherapy.

Background: The RAS-RAF-MEK-ERK pathway is commonly upregulated in human cancers. This is an open-label Phase 1 study of ASTX029, a dual-mechanism extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor, in subjects with relapsed/refractory solid tumors (NCT03520075). Methods: The primary objectives are to identify a maximum tolerated dose and/or recommended Phase 2 dose. ASTX029 was administered orally daily of 21-day cycles as powder-in-bottle (PiB, Cohort 1/10mg) and tablet formulation (beginning with Cohort 6/80 mg) under fed conditions, and as tablet formulation under fasting conditions (beginning with Cohort 8/40 mg). Dose escalation occurred according to a “3+3 design” based on dose-limiting toxicity (DLT) events. Disease response was evaluated according to RECIST v1.1 and exploratory indicators, including tumor variant allele frequency changes detected by cell-free DNA (cfDNA) quantitation. Results: 56 subjects were treated with at least one dose of ASTX029 in Phase 1A (dose escalation). Of 46 subjects with data, 35 (76%) had any RAS mutations and 4 (9%) had BRAF mutations; 1 subject had both. At the 200 mg dose level (Cohort 5, PiB/fed), one of six evaluable subjects developed a DLT (grade 3 maculopapular rash). At the 280 mg dose level (Cohort 12, tablet/fasting), two subjects experienced grade 2 central serous retinopathy adverse events (CSR AEs) within a few days of dosing. These were the only CSR AEs noted and one event was declared a DLT. Both subjects recovered to baseline within days of dose interruption. One cohort level below this dose was expanded (Cohort 11/200 mg, tablet/fasting); this dose level was deemed safe (without a DLT or grade ≥2 visual AE in 7 subjects) and was selected for Phase 1B dose expansion. Mean pharmacokinetic (PK) exposure was 151% of target exposure, which is defined as the level expected to have biological activity based on animal studies. The most frequent grade ≥2 AEs assessed as drug-related included nausea (4 subjects, grade 2) and transaminitis (4 subjects: 3 grade 2, 1 grade 3). The grade 3 transaminitis occurred in a subject with metastatic sarcoma involving the liver. There was one serious AE of malaise considered related to study drug. Two subjects, one with KRAS-G12A and BRAF-D549N non-small cell lung cancer (120 mg) and one with KRAS-G12D metastatic pancreatic cancer (200 mg), achieved partial responses (cycle 15/ongoing and cycle 3/ongoing, respectively). In 2 subjects with stable disease as the best response, longitudinal cfDNA sequencing showed a decrease of tumor variant allele frequencies after 2 cycles of ASTX029, followed by a return to baseline levels before disease progression. The most common reason for ASTX029 discontinuation was disease progression. Conclusions: This Phase 1A study of the ERK1/2 inhibitor ASTX029 has identified a dose level of 200 mg daily of a 21-day cycle for investigation in the Phase 1B portion of the study. Pharmacokinetic and pharmacodynamic data suggest target exposures are achieved with preliminary clinical activity. Citation Format: Patricia LoRusso, Drew Rasco, Geoffrey Shapiro, Filip Janku, Alain Mita, Nilofer Azad, Marcia Toguchi, Chris Hindley, Shannon Bradley, Danna Chan, Harold Keer, Kim-Hien Dao, Ryan J. Sullivan, Alexander Spira. A first-in-human, Phase 1 study of ASTX029, a dual-mechanism inhibitor of ERK1/2, in relapsed/refractory solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT108.

It is desirable to develop a fast method for quantification of melphalan due to its instability. Here we report a method for quantification of melphalan (MPL) in human plasma using a UPLC-PDA system. Briefly, 50µL plasma sample was mixed with 25µL internal standard (2500ng/mL acetylmelphalan in methanol) and 25µL 20% trichloroacetic acid, and centrifuged at 21,000g (15,000rpm) at 4°C for 3min. The supernatant (5µL) was injected onto an Acquity™ BEH C18 LC column (2.1 × 50mm, 1.7µm) and eluted with 25mM NH4AC (pH 4.7)-acetonitrile in a gradient mode at a flow rate of 0.6mL/min. The column kept at 40 ± 5°C and the autosampler kept at 4 ± 5°C. The detector set at 261nm, and sampling rate was 40points/sec. The retention times were typically 2.11min for melphalan and 2.38min for the internal standard. Total run time is 4min per sample. Calibration range was 100-40,000ng/mL. The lower limit of quantification was 100ng/mL. The method was validated based on the FDA guidelines, and applied to a clinical pharmacokinetic study in pediatric patients.

Background: ASTX660 is an oral, novel nonpeptidomimetic, small-molecule antagonist of cellular/X-linked inhibitors of apoptosis proteins (cIAP1/2 and XIAP). ASTX660 is currently being evaluated in a first-in-human phase 1–2 study in patients (pts) with advanced solid tumors or lymphoma (ClinicalTrials.gov NCT02503423). In the ongoing phase 2, ASTX660 has demonstrated preliminary evidence of clinical activity in the relapsed/refractory peripheral T-cell lymphoma (PTCL) and cutaneous T-cell lymphoma (CTCL) cohorts (Mehta et al, presented at the EHA Conference 2019, abs PS1073). Here, we report overall efficacy and safety data from the solid tumors (head and neck squamous cell carcinoma [HNSCC]; cervical carcinoma and other solid tumors) and lymphoma (diffuse large B-cell lymphoma [DLBCL], PTCL, CTCL,) phase 2 cohorts. Methods: Pts received treatment with ASTX660 orally at the RP2D 180mg/day on days 1 to 7, and 15 to 22 in a 28-day cycle. In the first stage 14 evaluable pts were enrolled in each of the 6 phase 2 cohorts with the option to expand the cohort if activity was observed. The primary endpoint was response rate as assessed by the investigator according to the Lugano criteria (DLBCL and PTCL), Global Assessment (CTCL), or RECIST 1.1 (solid tumors). Adverse events (AEs) were assessed per CTCAE V4.03. Results: As of June 4, 2019, a total of 107 pts have received ASTX660 in the solid tumors and lymphoma phase 2 cohorts (HNSCC n=14; DLBCL n=16; PTCL n=26; CTCL n=23; cervical carcinoma n=14; other solid tumors n= 14). Median age (range) was 61 (23-84) years and median number (range) of prior anticancer regimens was 3 (0-12). Among all pts, the most common related AEs of any grade (≥ 10%) were rash (35%), lipase elevation (34%), amylase elevation (29%), diarrhea (14%), fatigue (14%), AST elevation (13%), nausea (13%), and anemia (11%). Related AEs ≥ Grade 3 occurring in ≥ 5% of pts were rash (18%), lipase elevation (16%) and amylase elevation (9%). As of 4 June 2019, 86 pts (80%) discontinued study treatment: 64 (60%) due to progressive disease, 13 (12%) due to AE, 4 (4%) due to death, 4 (4%) due to withdrawal by participant and 1 (1%) for investigator’s decision. At the time of analysis, the ORR was 36% in the PTCL cohort and 15% in the CTCL cohort. One PR was reported in a pt with metastatic melanoma after 12 cycles of treatment. No objective responses were reported in the HNSCC, DLBCL or cervical cohorts. Accrual in the PTCL and CTCL continues; updated efficacy and safety data will be presented at the meeting. Conclusion: In the phase 2 part of the study ASTX660 monotherapy has demonstrated a manageable safety profile and encouraging activity in PTCL and CTCL warranting cohort expansion. Future plans include evaluation of ASXT660 both as mono- or combination therapy in selected malignancies. Citation Format: Antoine Hollebecque, Sarit Assouline, Felipe Samaniego, Benoit You, Francine Foss, Anca Prica, Sarah W. Gordon, Marc Webster, Martin JS. Dyer, Dima El-Sharkawi, Geoffrey I. Shapiro, Lisa Nabell, Sarah P. Blagden, John Lister, Susanna V. Ulahannan, Yijun Sun, Danna Chan, Roberta Ferraldeschi, Monica Mita. Preliminary results of ASTX660, a novel non-peptidomimetic cIAP1/2 and XIAP antagonist, in 107 patients with solid tumors or lymphoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A072. doi:10.1158/1535-7163.TARG-19-A072

A prospective multicenter study was conducted to characterize the pharmacokinetics (PK) and pharmacodynamics (PD) of fludarabine plasma (f-ara-a) and intracellular triphosphate (f-ara-ATP) in children undergoing hematopoietic cell transplantation (HCT) and receiving fludarabine with conditioning. Plasma and peripheral blood mononuclear cells (PBMCs) were collected over the course of therapy for quantitation of f-ara-a and f-ara-ATP. Nonlinear mixed-effects modeling was used to develop the PK model, including identification of covariates impacting drug disposition. Data from a total of 133 children (median age, 5 years; range, .2 to 17.9) undergoing HCT for a variety of malignant and nonmalignant disorders were available for PK-PD modeling. The implementation of allometric scaling of PK parameters alone was insufficient to describe drug clearance, particularly in very young children. Renal impairment was predicted to increase drug exposure across all ages. The rate of f-ara-a entry into PBMCs (expressed in pmoles per million cells) decreased over the course of therapy, resulting in 78% lower f-ara-ATP after the fourth dose (1.7 pmoles/million cells [range, .2 to 7.2]) compared with first dose (7.9 pmoles/million cells [range, .7 to 18.2]). The overall incidence of treatment-related mortality (TRM) was low at 3% and 8% at days 60 and 360, respectively, and no association with f-ara-a exposure and TRM was found. In the setting of malignancy, disease-free survival was highest at 1 year after HCT in subjects achieving a systemic f-ara-a cumulative area under the curve (cAUC) greater than 15 mg*hour/L compared to patients with a cAUC less than 15 mg*hour/L (82.6% versus 52.8% P = .04). These results suggest that individualized model-based dosing of fludarabine in infants and young children may reduce morbidity and mortality through improved rates of disease-free survival and limiting drug-related toxicity. ClinicalTrials.gov Identifier: NCT01316549

Cytoplasmic dynein is a dimeric motor that transports intracellular cargoes towards the minus end of microtubules (MTs). In contrast to other processive motors, stepping of the dynein motor domains (heads) is not precisely coordinated. Therefore, the mechanism of dynein processivity remains unclear. Here, by engineering the mechanical and catalytic properties of the motor, we show that dynein processivity minimally requires a single active head and a second inert MT-binding domain. Processivity arises from a high ratio of MT-bound to unbound time, and not from interhead communication. In addition, nucleotide-dependent microtubule release is gated by tension on the linker domain. Intramolecular tension sensing is observed in dynein's stepping motion at high interhead separations. On the basis of these results, we propose a quantitative model for the stepping characteristics of dynein and its response to chemical and mechanical perturbation.

Cytoplasmic dynein is a processive minus end directed microtubule motor involved in a wide range of cellular functions. By studying how dynein monomers respond to load in an optical trapping assay, we discovered a force dependent gating mechanism that prevents ATP driven release of dynein from the microtubule. Our results show that tension on dynein's linker domain causes the microtubule release rate of the motor to be insensitive to ATP, while dynein motors with an unloaded linker release at a much faster rate at high ATP concentrations. We found that dynein monomers preferentially release when force is applied towards the minus end of the microtubule rather than the plus end. This strong asymmetry is observed even in the absence of dynein's ATPase ring, implying it is an intrinsic property of the stalk and microtubule binding domain. To test what role these phenomena play in dynein motility, we studied ATPase mutants with altered translocation velocities and measured their force dependent release kinetics. On the basis of these results we developed a minimal model of dynein motility and show how intramolecular tension, powerstroke and force dependent release are main determinants of dynein's motility properties and directionality.

Cytoplasmic dynein is a molecular motor responsible for minus-end directed transport along microtubules. In contrast to kinesins and myosins, the detailed mechanism of dynein processivity and force generation remains unclear. Dynein’s structure and evolutionary origin are different from these motors, suggesting unique mechanistic features. In this work, we engineered novel dynein constructs with altered mechanical, chemical and geometric properties to test the roles of rigid linkage between monomers, interaction between the ATPase rings and the proposed linker swing mechanism in maintaining dynein processivity. We found that a rigid linkage between monomers and dimerization through the N-terminal tail domains are not essential for dynein processivity. Instead, processivity minimally requires the linker domain of one monomer with an active ATPase ring to be attached to a partner monomer, which can be replaced by an inert protein retaining the microtubule-binding domain. To understand how one active head could provide motility to an inactive partner, we quantified the force-dependent microtubule release rates of dynein monomers. A clear directional asymmetry for detachment was observed, with significantly faster release towards the minus-end, providing insight into dynein’s directionality. These results led us to a detailed mechanistic model of dynein processivity, directionality and force generation.