The Fanconi anemia (FA)-BRCA pathway mediates repair of DNA interstrand crosslinks. The FA core complex, a multi-subunit ubiquitin ligase, participates in the detection of DNA lesions and monoubiquitinates two downstream FA proteins, FANCD2 and FANCI (or the ID complex). However, the regulation of the FA core complex itself is poorly understood. Here we show that the FA core complex proteins are recruited to sites of DNA damage and form nuclear foci in S and G2 phases of the cell cycle. ATR kinase activity, an intact FA core complex and FANCM-FAAP24 were crucial for this recruitment. Surprisingly, FANCI, but not its partner FANCD2, was needed for efficient FA core complex foci formation. Monoubiquitination or ATR-dependent phosphorylation of FANCI were not required for the FA core complex recruitment, but FANCI deubiquitination by USP1 was. Additionally, BRCA1 was required for efficient FA core complex foci formation. These findings indicate that FANCI functions upstream of FA core complex recruitment independently of FANCD2, and alter the current view of the FA-BRCA pathway.
SUMMARY Repair of interstrand crosslinks by the Fanconi anemia (FA) pathway requires both the monoubiquitination and de-ubiquitination of the FANCI/FANCD2 (FANCI/D2) complex. In the standing model, the phosphorylation of six sites in the FANCI S/TQ cluster domain occurs upstream of, and promotes, FANCI/D2 monoubiquitination. We generated phospho-specific antibodies against three different S/TQ cluster sites (Serines 556, 559, and 565) on human FANCI and found that, in contrast to the standing model, distinct FANCI sites were phosphorylated either predominantly upstream (ubiquitination-independent; Serine 556) or downstream (ubiquitination-linked; Serines 559 and 565) of FANCI/D2 monoubiquitination. Ubiquitination-linked FANCI phosphorylation inhibited FANCD2 de-ubiquitination and bypassed the need to de-ubiquitinate FANCD2 to achieve effective interstrand crosslink repair. USP1 depletion suppressed ubiquitination-linked FANCI phosphorylation despite increasing FANCI/D2 monoubiquitination, providing an explanation of why FANCD2 de-ubiquitination is important for function of the FA pathway. Our work results in a refined model of how FANCI phosphorylation activates the FANCI/D2 complex.
Fanconi anemia (FA), the most common form of inherited bone marrow failure, predisposes to leukemia and solid tumors. FA is caused by the genetic disruption of a cellular pathway that repairs DNA interstrand crosslinks. The impaired function of this pathway, and the genetic instability that results, is considered the main pathogenic mechanism behind this disease. The identification of breast cancer susceptibility genes (for example, BRCA1/FANCS and BRCA2/FANCD1) as being major players in the FA pathway has led to a surge in molecular studies, resulting in the concept of the FA-BRCA pathway. In this review, we discuss recent advances in the molecular pathogenesis of FA from three viewpoints: i) new FA genes, ii) modifier pathways that influence the cellular and clinical phenotypes of FA and iii) non-canonical functions of FA genes that may drive disease progression independently of deficient DNA repair. Potential therapeutic approaches for FA that are relevant to each will also be proposed.
BRCA1 C-terminal domains are found in a specialized group of 23 proteins that function in the DNA damage response to protect genomic integrity. C-terminal domain phosphatase 1 (CTDP1) is the only phosphatase with a BRCA1 C-terminal domain in the human proteome, yet direct participation in the DNA damage response has not been reported. Examination of the CTDP1 BRCA1 C-terminal domain-specific protein interaction network revealed 103 high confidence interactions enriched in DNA damage response proteins, including FANCA and FANCI that are central to the Fanconi anemia DNA repair pathway necessary for the resolution of DNA interstrand crosslink damage. CTDP1 expression promotes DNA damage-induced FANCA and FANCD2 foci formation and enhances homologous recombination repair efficiency. CTDP1 was found to regulate multiple aspects of FANCI activity, including chromatin localization, interaction with γ-H2AX, and SQ motif phosphorylations. Knockdown of CTDP1 increases MCF-10A sensitivity to DNA interstrand crosslinks and double-strand breaks, but not ultraviolet radiation. In addition, CTDP1 knockdown impairs in vitro and in vivo growth of breast cancer cell lines. These results elucidate the molecular functions of CTDP1 in Fanconi anemia interstrand crosslink repair and identify this protein as a potential target for breast cancer therapy.
The MYC oncogene induces both cell proliferation and apoptosis. The apoptotic function of MYC is thought to inhibit carcinogenesis; thus, when disrupted, tumorigenic potential is increased. Both MYC and transforming growth factor alpha (TGFalpha) are commonly over-expressed in hepatocellular carcinomas, and transgenic mice expressing these genes rapidly develop tumors via the suppression of MYC-induced apoptosis by the growth factor. However, the nature of the interactions between MYC and TGFalpha are not well understood. Specifically, it is unclear whether TGFalpha acts only as an anti-apoptotic factor in its interactions with MYC or whether it has substantial effects on cell growth. We investigated whether TGFalpha can provide additional mitogenic signals if it is not required to act as an anti-apoptotic factor. We demonstrate that expression of MYC and TGFalpha in liver progenitor cells (known as oval cells) results in enhanced cell proliferation in culture and the generation of poorly differentiated tumors after inoculation into nude mice. We further demonstrate that while the apoptosis-deficient T58A and S71F alleles of MYC retain their ability to promote oval cell proliferation, they have opposite growth interactions with TGFalpha. The T58A allele has a stimulatory effect on both proliferation and tumorigenicity. In contrast, co-expression of the S71F allele reduces proliferation and slows tumor development. We conclude that the tumorigenic growth effects of MYC in TGFalpha-expressing liver progenitor cells are not solely dependent on its apoptotic activity.
Background: Background: CPX-351 (Europe: Vyxeos ® liposomal; United States: Vyxeos ® ) is a dual-drug liposomal encapsulation of daunorubicin and cytarabine in a synergistic 1:5 molar ratio. CPX-351 is approved for newly diagnosed, therapyrelated acute myeloid leukemia (AML) or AML with myelodysplasia-related changes in adults in Europe and patients aged ≥1 year in the United States who are candidates for intensive chemotherapy (IC). However, the appropriate dosage of CPX-351 in patients unfit for IC may be different from the label dosage. Venetoclax (VEN; BCL-2 inhibitor) + low-dose cytarabine has demonstrated efficacy in unfit patients with AML, and drug synergism/additivity in preclinical studies provided a rationale for combining CPX-351 + VEN clinically. Aims:Aims: Our study evaluates the safety and efficacy of lower-intensity CPX-351 + VEN in adults with newly diagnosed AML who are unfit for IC. Methods:Methods: This is an ongoing, open-label, phase 1b study (NCT04038437). Patients who achieve at least partial remission after 1 or 2 cycles may receive up to 4 similar cycles in the dose-exploration phase (DEP) or up to 8 similar cycles in the expansion phase (EP). Patients are assessed for response (morphology, measurable residual disease [MRD]) and monitored for safety and survival. Results:Results: The data include 31 patients enrolled by 15 September 2021, with a data cutoff of 2 December 2021: 4 patients in the DEP at dose level 1 (CPX-351 20 units/m 2 on Days 1 and 3 + VEN 400 mg on Days 2 to 21 of each cycle), 7 patients in the DEP at dose level 2 (CPX-351 40 units/m 2 + VEN 400 mg), and a total of 20 patients in the DEP and EP at dose level 1b (CPX-351 30 units/m 2 + VEN 400 mg), which was established as the recommended phase 2 dose. Patients were considered unfit for IC based on age ≥75 years (n=15) or health (Eastern Cooperative Oncology Group performance status of 2 to 3 and/or comorbidities [n=16]). Median age was 74 years (range: 60, 90); 65% were male; 77% had de novo AML; 58% had poor-risk disease; and 23% had a TP53 mutation.Nonhematologic treatment-emergent adverse events (TEAEs) in ≥20% of patients were diarrhea (26%), cough (23%), dyspnea (23%), and nausea (23%). Hematologic grade ≥3 TEAEs were reported in 17 (55%) patients; no nonhematologic grade ≥3 TEAE was reported in >10% of patients. There were no deaths by Day 30; mortality at Day 60 was 13%, with deaths due to myocardial infarction unrelated to therapy (n=1), worsening lung infection (n=1), and disease progression (n=2). Median (interquartile range) recovery times were 30 days (22, 34.5) to neutrophils ≥500/μL and 21 days (21, 27) to platelets ≥50,000/μL. Complete remission (CR) or CR with incomplete neutrophil or platelet recovery (CRi) was achieved by 16/28 (57%) patients with an evaluable remission assessment. All 16 of these patients achieved remission (CR or CRi) after the first treatment cycle. MRD negativity was achieved by 12/16 (75%) patients with CR or CRi, primarily after Cycle 1 (Cycle 1: n=8; Cycle 2: n=2; Cycle 3: n=1;...
Introduction: CPX-351 (Vyxeos®; daunorubicin and cytarabine liposome for injection), a dual-drug liposomal encapsulation of daunorubicin and cytarabine in a synergistic 1:5 molar drug ratio, has been approved by the US FDA and EMA for the treatment of adults with newly diagnosed therapy-related AML or AML with myelodysplasia-related changes. A randomized phase 3 study that evaluated patients 60 to 75 years of age with newly diagnosed high-risk/secondary AML provided the basis for approval and demonstrated that induction followed by consolidation with CPX-351 significantly improved overall survival and remission rates versus conventional 7+3, with a comparable safety profile. Several targeted therapies have demonstrated efficacy when added to cytotoxic therapy for the treatment of patients with previously untreated AML, including venetoclax (Venclexta®/Venclyxto®; a B-cell leukemia/lymphoma-2 [BCL-2] inhibitor), midostaurin (Rydapt®; a FMS-related tyrosine kinase 3 [FLT3] inhibitor), and enasidenib (Idhifa®; an isocitrate dehydrogenase 2 [IDH2] inhibitor). Preclinical data indicate synergistic activity may be achieved with CPX-351 in combination with venetoclax or midostaurin. Results of these studies suggest a rationale for the combination of targeted therapies using CPX-351 as a chemotherapy backbone. Study Design and Methods: V-FAST (Vyxeos - First Phase Assessment with Targeted Agents) is an open-label, multicenter, multi-arm, nonrandomized, phase 1b master trial (ClinicalTrials.gov #NCT04075747) to evaluate CPX-351 in combination with targeted agents (venetoclax, midostaurin, or enasidenib) in adults with previously untreated AML who are considered fit for intensive chemotherapy. As V-FAST is a master trial, it is designed to permit the expedited incorporation of CPX-351 combinations with other targeted agents into the study as it proceeds, thus ensuring a timely investigation of novel combinations moving forward. Key eligibility criteria are shown in Table 1. The primary study endpoints are to establish the recommended phase 2 dose (RP2D) and safety of each combination regimen. Secondary endpoints include remission rates (via morphologic assessment), bone marrow measurable residual disease status, and pharmacokinetics. Exploratory endpoints include duration of remission, overall survival, event-free survival, and the proportion of patients proceeding to hematopoietic cell transplantation. Patients will be monitored for safety until 1 month following the end of treatment and survival for up to 2 years following the first administration of treatment. Cytogenetic and/or molecular testing will determine which treatment arm each patient is assigned to (ie, no FLT3 or IDH2 mutations: CPX-351 plus venetoclax; FLT3 mutation: CPX-351 plus midostaurin; IDH2 mutation: CPX-351 plus enasidenib). For each combination, a dose-exploration phase (standard 3+3 design; up to 12 patients/combination) will employ dose de-escalation or escalation of CPX-351 and/or the targeted agent based on the occurrence of dose-limiting toxicities to determine a RP2D and evaluate the safety of the combination. The initial cohort for each arm will be treated with the dosing described in Table 2. In the subsequent expansion phase for each combination, an additional 20 patients will be treated to confirm the RP2D, further evaluate safety, and provide an initial assessment of efficacy. In both study phases, CPX-351 will be administered intravenously by 90-minute infusion on Days 1, 3, and 5; targeted therapies will be administered per a standard route for each agent. Patients may receive 1 to 2 induction cycles of combination therapy. Those achieving remission may receive up to 2 consolidation cycles with CPX-351 plus the targeted agent received during induction; hematopoietic stem cell transplantation is permitted in place of or following chemotherapy consolidation at the discretion of the treating physician. This study is ongoing and actively enrolling patients. Disclosures Lin: Celgene: Research Funding; Aptevo: Research Funding; Abbvie: Research Funding; Astellas Pharma: Research Funding; Ono Pharmaceutical: Research Funding; Mateon Therapeutics: Research Funding; Prescient Therapeutics: Research Funding; Pfizer: Research Funding; Celyad: Research Funding; Genetech-Roche: Research Funding; Gilead Sciences: Research Funding; Incyte: Research Funding; Trovagene: Research Funding; Tolero Pharmaceuticals: Research Funding; Seattle Genetics: Research Funding; Bio-Path Holdings: Research Funding; Jazz: Research Funding. Mannis:AbbVie, Agios, Bristol-Myers Squibb, Genentech: Consultancy; Glycomimetics, Forty Seven, Inc, Jazz Pharmaceuticals: Research Funding. Erba:Glycomimetics: Other: member of Scientific Steering Committee; AbbVie, Daiichi Sankyo, Forma, ImmunoGen, Jazz Pharmaceuticals, MacroGenics, Novartis, PTC: Research Funding; AbbVie, Agios, Celgene, Incyte, Jazz Pharmaceuticals, and Novartis: Speakers Bureau; AbbVie, Agios, Amgen, Astellas, Celgene, Daiichi Sankyo, Glycomimetics, ImmunoGen, Incyte, Jazz Pharmaceuticals, MacroGenics, Novartis, and Pfizer: Consultancy; Celgene: Other: chair of the Scientific Steering Committee; Covance (AbbVie): Other: chair of the Independent Review Committee. Levis:Daiichi-Sankyo: Honoraria; FujiFilm: Honoraria, Research Funding; Astellas: Honoraria, Research Funding; Amgen: Honoraria; Menarini: Honoraria. Zou:Jazz Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Faderl:Jazz Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Cheung:Jazz Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Pullarkat:Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie, Inc.: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Genetech: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Dova: Consultancy, Honoraria; Servier: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. OffLabel Disclosure: This study will explore CPX-351 in combination with targeted agents for the treatment of adults with AML.
7026 Background: CPX-351 (US: Vyxeos; EU: Vyxeos Liposomal), a dual-drug liposomal encapsulation of daunorubicin and cytarabine in a synergistic 1:5 molar drug ratio, is approved by the US FDA and EMA for adults with newly diagnosed t-AML or AML with myelodysplasia-related changes. Preclinical data suggest CPX-351 may exert synergistic activity when combined with agents such as the BCL-2 inhibitor venetoclax (VEN) or FLT3 inhibitor midostaurin (MIDO). Methods: V-FAST (Vyxeos – First Phase Assessment With Targeted Agents) is an open-label, multicenter, phase 1b master trial (NCT04075747) to evaluate safety and establish the recommended phase 2 dose (RP2D) of CPX-351 combined with targeted agents in patients (pts) aged 18-75 y with untreated AML who are fit for intensive chemotherapy. The study includes a dose-exploration phase (3+3 design) and subsequent expansion phase. Pts received CPX-351 (dose level 1 for first induction [DL1]: 100 units/m2 on Days 1, 3, and 5) plus VEN (Arm A; DL1: 400 mg on Days 1-14), MIDO (Arm B; DL1: 50 mg BID on Days 8-21), or the IDH2 inhibitor enasidenib ([ENA] Arm C; DL1: 100 mg on Days 8-28) based on mutation testing. Results: Among 21 pts with available data enrolled by 11/06/20 (24 pts enrolled total; data cut-off: 01/19/21), the median age was 54 y (range: 35, 69). In Arm A (n = 17), 11 (65%) pts had de novo AML, 5 (29%) had an antecedent hematologic disorder (2 [12%] had myelofibrosis), and 2 (12%) had t-AML; 12 (71%) had adverse-risk AML; and 6 (35%) had mutated TP53. In Arms B (n = 3) and C (n = 1), all pts had intermediate-risk de novo AML. DL1 was the RP2D in Arms A and B; the RP2D in Arm C is still under investigation. In Arm A, 1/6 pts in the dose-exploration phase had 2 dose-limiting toxicities (DLTs) of grade 4 neutropenia and thrombocytopenia that extended beyond 49 days; no DLTs have occurred for Arms B and C. The combinations exhibited manageable safety profiles (Table). Of pts with available response data, complete remission (CR) or CR with incomplete platelet or neutrophil recovery was achieved by 6/14 (43%) pts in Arm A, including 4 (29%) with CR. All pts in Arms B and C achieved CR. Conclusions: These preliminary results suggest CPX-351 can be combined with VEN and MIDO with manageable toxicities in newly diagnosed AML pts, with DL1 determined to be the RP2D. The study is ongoing and actively enrolling pts; updated results will be presented at the meeting. Clinical trial information: NCT04075747. [Table: see text]
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