ALBERT

Description: Introduction: Smoking is a potential risk factor for the development of non-Hodgkin lymphoma (NHL), and prior studies have reported inferior survival in tobacco users with certain subtypes of the disease (Taborelli et al, BMC Cancer, 2017; Ollberding et al, Br J Haematol, 2013). For instance, tobacco smokers with NHL had an inferior overall survival (OS) compared to non-smokers in a series of 471 patients who were managed up front with either chemotherapy (68%), radiation (27%), or observation, and this appeared to be most pronounced in patients with follicular lymphoma and in those with a 20+ pack year smoking history (Geyer et al, Cancer, 2010). The impact of tobacco use on survival specifically in patients with mantle cell lymphoma (MCL) has not been well studied. We conducted a multicenter study in MCL and evaluated the prognostic impact of tobacco use. Methods: We included patients with MCL from 12 sites who were ≥18 years old and for whom smoking status was known at the time of diagnosis. Cases were evaluated for reported smoking status at the time of diagnosis (active smoker, prior smoker, or never smoker) and standard baseline clinical prognostic data were obtained for each patient. Descriptive statistics were generated for these characteristics and were then compared across smoking status using chi-squared tests, Fisher's exact tests, or ANOVA, where appropriate. Overall survival (OS) and progression free survival (PFS) were estimated using the Kaplan-Meier method, and were compared using log-rank tests. Results: Of 946 included patients, 456 (48.2%) reported never using tobacco, 360 (38.7%) reported prior tobacco use, and 130 (13.7%) reported active tobacco use at the time of diagnosis. Median age was 59 in the active smoker group, 65 in prior smokers, and 61 in never smokers (p 〈 0.001). Any major medical comorbidity (defined as the presence of CAD, CHF, diabetes, CKD, ESRD, COPD, DVT, prior malignancy, or cirrhosis) was present in 59 (45.4%) of the active smokers, 143 (39.7%) of the prior smokers, and 140 (30.7%) of the never smokers (p = 0.002). Intensive induction regimens were used in 58.2% of active smokers, 47.2% of prior smokers, and 58.4% of never smokers (p=0.007). There were no significant differences between groups in regards to sex, race, ECOG performance status, Ann Arbor stage, time to first treatment, and use of auto transplant in first remission. Patients with no prior history of tobacco use were less likely to have a high risk MIPI score at diagnosis (26% high risk) compared to prior smokers (39.5%) and active smokers (32.5%, p=0.019). With a median follow up of 3.5 years after diagnosis, there was no significant difference between the 3 groups with regards to PFS or OS (Figure 1). Five-year OS in the never smoker group was 79.8% (95% CI: 74.8%, 83.9%) vs 75.1% (64.5%, 82.9%) in the active smoker group, and 80.6% (74.6%, 85.3%) in the prior smoker group (log rank p = 0.4079). Five- year progression free survival was 50.4% (44.6%, 56.0%) in the never smoker group, 42.5% (32.2%, 52.5%) in the active smoker group, and 50.2% (43.5%, 56.6%) in the prior smoker group (log rank p= 0.3595). Conclusions: Our data suggest that active or prior smoking does not significantly impact OS or PFS in patients with MCL. This study is limited by the fact that amount of current or former tobacco use was not available and it is not known how many current tobacco users ultimately stopped smoking during the course of their treatment. Future studies should incorporate more specific information regarding smoking history including pack-years and time between discontinuation of tobacco use and date of diagnosis. While tobacco use and other modifiable cardiovascular risk factors should be addressed as appropriate for all patients with MCL, current and former tobacco users can still achieve prolonged PFS and OS and may be candidates for intensive treatments after consideration of their other comorbidities and disease-specific risk factors. Disclosures Calzada: Seattle Genetics: Research Funding. Kolla:Amgen: Equity Ownership. Bachanova:Gamida Cell: Research Funding; GT Biopharma: Research Funding; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees; Incyte: Research Funding; Celgene: Research Funding; Novartis: Research Funding; Kite: Membership on an entity's Board of Directors or advisory committees. Gerson:Seattle Genetics: Consultancy; Abbvie: Consultancy; Pharmacyclics: Consultancy. Barta:Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Mundipharma: Honoraria; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Takeda: Research Funding; Merck: Research Funding; Mundipharma: Honoraria; Bayer: Consultancy, Research Funding; Seattle Genetics: Honoraria, Research Funding. Danilov:Celgene: Consultancy; Abbvie: Consultancy; TG Therapeutics: Consultancy; Bayer Oncology: Consultancy, Research Funding; Gilead Sciences: Consultancy, Research Funding; Janssen: Consultancy; AstraZeneca: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Aptose Biosciences: Research Funding; Bristol-Meyers Squibb: Research Funding; MEI: Research Funding; Pharmacyclics: Consultancy; Verastem Oncology: Consultancy, Other: Travel Reimbursement , Research Funding; Curis: Consultancy; Takeda Oncology: Research Funding; Seattle Genetics: Consultancy. Grover:Seattle Genetics: Consultancy. Karmali:Astrazeneca: Speakers Bureau; Takeda, BMS: Other: Research Funding to Institution; Gilead/Kite; Juno/Celgene: Consultancy, Speakers Bureau. Hill:Seattle Genetics: Consultancy, Honoraria; Takeda: Research Funding; Amgen: Research Funding; TG therapeutics: Research Funding; AstraZeneca: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celegene: Consultancy, Honoraria, Research Funding; Genentech: Consultancy, Research Funding; Kite: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Ghosh:Pharmacyclics: Consultancy, Research Funding, Speakers Bureau; Seattle Genetics: Consultancy, Speakers Bureau; Genentech: Research Funding; Celgene: Consultancy, Research Funding, Speakers Bureau; Forty Seven Inc: Research Funding; Gilead/Kite: Consultancy, Speakers Bureau; Spectrum: Consultancy, Speakers Bureau; AbbVie: Consultancy, Speakers Bureau; T G Therapeutics: Consultancy, Research Funding; Astra Zeneca: Speakers Bureau. Park:BMS: Consultancy, Research Funding; Rafael Pharma: Membership on an entity's Board of Directors or advisory committees; G1 Therapeutics: Consultancy; Teva: Consultancy, Research Funding; Gilead: Speakers Bureau; Seattle Genetics: Research Funding, Speakers Bureau. Epperla:Pharmacyclics: Honoraria; Verastem Oncology: Speakers Bureau. Hamadani:Pharmacyclics: Consultancy; ADC Therapeutics: Consultancy, Research Funding; Merck: Research Funding; Celgene: Consultancy; Janssen: Consultancy; Medimmune: Consultancy, Research Funding; Sanofi Genzyme: Research Funding, Speakers Bureau; Otsuka: Research Funding; Takeda: Research Funding. Kahl:TG Therapeutics: Consultancy; BeiGene: Consultancy; Seattle Genetics: Consultancy; ADC Therapeutics: Consultancy, Research Funding. Martin:Janssen: Consultancy; Sandoz: Consultancy; I-MAB: Consultancy; Teneobio: Consultancy; Celgene: Consultancy; Karyopharm: Consultancy. Flowers:Karyopharm: Consultancy; Denovo Biopharma: Consultancy; Burroughs Wellcome Fund: Research Funding; AbbVie: Consultancy, Research Funding; Gilead: Consultancy, Research Funding; Spectrum: Consultancy; AstraZeneca: Consultancy; Pharmacyclics/Janssen: Consultancy, Research Funding; Bayer: Consultancy; Acerta: Research Funding; Genentech, Inc./F. Hoffmann-La Roche Ltd: Consultancy, Research Funding; Optimum Rx: Consultancy; Millenium/Takeda: Research Funding; Eastern Cooperative Oncology Group: Research Funding; National Cancer Institute: Research Funding; V Foundation: Research Funding; BeiGene: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; TG Therapeutics: Research Funding. Cohen:Genentech, Inc.: Consultancy, Research Funding; Janssen Pharmaceuticals: Consultancy; Takeda Pharmaceuticals North America, Inc.: Research Funding; Gilead/Kite: Consultancy; LAM Therapeutics: Research Funding; UNUM: Research Funding; Hutchison: Research Funding; Astra Zeneca: Research Funding; Lymphoma Research Foundation: Research Funding; ASH: Research Funding; Seattle Genetics, Inc.: Consultancy, Research Funding; Bristol-Meyers Squibb Company: Research Funding.

Description: Background: Infusion of chimeric antigen receptor modified T cells targeting the CD30 molecule and encoding the CD28 endodomain (CD30.CAR-Ts) in the absence of lymphodepleting chemotherapy has been shown to be safe with responses seen in patients (pts) with relapsed/refractory (r/r) CD30+ lymphomas (Ramos et al., JCI 2017). We present here the results of a phase 1b/2 trial of CD30.CAR-Ts administered after lymphodepleting chemotherapy in pts with r/r CD30+ Hodgkin (HL) and Non-Hodgkin lymphoma (NHL). Methods: The primary objective of the phase 1b portion of the study was to determine the recommended phase 2 dose level (RP2DL) of CD30.CAR-Ts using a standard 3+3 design. Two dose levels were tested: 1 x 108 CAR-Ts/m2 (DL1) and 2 x 108 CAR-Ts/m2 (DL2). For lymphodepletion, the first 8 pts (including the first 3 pts treated at DL1) received 2 days of bendamustine (benda) 90 mg/m2, while the 10 remaining pts received 3 days of benda 70 mg/m2 and fludarabine (flu) 30 mg/m2, except for one pt who received only 1 day of benda and flu due to possible benda toxicity. Inclusion criteria were age ≥ 18 years, CD30+ disease, and r/r HL or NHL having failed ≥2 prior therapies. Results: At the time of data cut off (7/1/2018), 18 pts with a median age of 40.5 years (range: 23-70) had received CD30.CAR-Ts and undergone response assessment. Sixteen pts had HL, 1 had enteropathy associated T cell lymphoma, and 1 had Sezary syndrome. All pts were heavily pre-treated with a median of 8.5 prior therapies (range: 4-17). All pts had received prior brentuximab vedotin and 13 had prior checkpoint inhibitor therapy. Fourteen pts had prior autologous stem cell transplant (SCT) and 7 had prior allogeneic SCT. Treatment was well tolerated with no dose limiting toxicities; as a consequence, the highest dose level of CAR-T cells (2 x 108 CAR-Ts/m2) was given as the RP2DL. Three pts developed cytokine release syndrome (CRS) (grade 1: 2 pts and grade 2: 1 pt). Grade 1 CRS resolved spontaneously, while the pt with grade 2 CRS responded to tocilizumab. No neurotoxicity was observed. Out of the 18 pts, 4 were in a complete response (CR) before infusion due to bridging chemotherapy and remained disease free at 6 wk follow up. Two of these pts have since relapsed with PFS of 3.8 months and 11.9 months while the other 2 pts are still in CR after 1 year of follow up. The 14 pts with evidence of disease pre-lymphodepletion were included in efficacy analysis. Of these 14 pts, 6 had a CR (43%, all in the benda/flu cohort), 1 had partial response (7%), 2 had stable disease (14%) and 5 had progressive disease (35%) at disease assessment. No responses occurred in the 3 pts treated at DL1. At median follow up of 138 days, the median PFS was 129 days. The median PFS for the 3 evaluable pts who received benda at DL1 was 55 days vs 172 days for the 9 pts who received benda/flu at DL2 (p = 0.039). The median PFS for the 2 evaluable pts at DL2 who received benda lymphodepletion was 85.5 days but this was not included in the comparison due to small sample size. Two out of 14 evaluable pts remain in CR at 1 year. Using PCR on peripheral blood, CD30.CAR-Ts were found to be increased in the circulation of all pts, peaking at wk 2 post infusion, with increasing CAR-T cells in pts receiving greater number of CAR-T cells or more robust lymphodepletion (3.4x103 ± 2.9x103 copies/ug of DNA for DL1-beda vs. 61x103 ± 41x103 for DL2-benda vs. 59x103 ± 22x103 for benda/flu). These differences were confirmed by flow cytometry (CD3+CAR+ cells = 13%±9% for DL1-benda vs 21%±10% for DL2-benda vs 35%±8% for benda/flu). Persistence was also related to dose level and lymphodepletion (0.06x103 ± 0.01x103 vs 0.44x103 ± 0.41x103 vs 28x103 ± 15x103/ug of DNA at wk 4 for DL1-benda, DL2-benda, and benda/flu, respectively). Although both lymphodepletion regimens reduced the lymphocyte counts, only the combination of benda/flu was found to have a significant increase in IL-15 and IL-7 cytokines (13 fold, p

Description: The Fanconi anemia (FA) complementation group C (FAC) protein gene encodes a cytoplasmic protein with a predicted Mrof 63,000. The protein's function is unknown, but it has been hypothesized that it either mediates resistance to DNA cross-linking agents or facilitates repair after exposure to such factors. The protein also plays a permissive role in the growth of colony-forming unit–granulocyte/macrophage (CFU-GM), burst-forming unit–erythroid (BFU-E), and CFU-erythroid (CFU-E). Attributing a specific function to this protein requires an understanding of its intracellular location. Recognizing that prior study has established the functional importance of its cytoplasmic location, we tested the hypothesis that FAC protein can also be found in the nucleus. Purified recombinant Escherichia coli–derived FAC antigens were used to create antisera able to specifically identify an Mr = 58,000 protein in lysates from human Epstein-Barr virus (EBV)-transformed cell lines by immunoblot analysis. Subcellular fractionation of the cell lysates followed by immunoblot analysis revealed that the majority of the FAC protein was cytoplasmic, as reported previously; however, approximately 10% of FAC protein was reproducibly detected in nuclear fractions. These results were reproducible by two different fractionation methods, and included markers to control for contamination of nuclear fractions by cytoplasmic proteins. Moreover, confocal image analysis of human 293 cells engineered to express FAC clearly demonstrated that FAC protein is located in both cytoplasmic and nuclear compartments, consistent with data obtained from fractionation of the FA cell lines. Finally, complementation of the FAC defect using retroviral-mediated gene transfer resulted in a substantial increase in nuclear FAC protein. Therefore, while cytoplasmic localization of this protein appears to be functionally important, it may also exert some essential nuclear function.

Description: Recent work has clarified a role for aldehyde dehydrogenases in protecting Fanconi anemia (FA) hematopoietic stem cells (HSC) and indicates that the increase in endogenous aldehydes that attends the genetic loss of ALDH function is sufficient to induce bone marrow failure in this disease. Studies in many laboratories have also documented that: (1) exposure of FA HSC to inflammatory cytokines suppresses stem cell self-replication and, in vivo, exhausts the stem cell pool and (2) FA macrophages exposed to specific toll-like receptor (TLR) ligands overproduce the very inflammatory cytokines that suppress the HSC pool. Given that aldehydes form adducts with and, in some cases, activate signaling proteins involved in cytokine production, we tested the hypothesis that overproduction of inflammatory cytokines by FA macrophages results from a loss of FA protein-dependent ALDH function. We treated THP-1 human monocytic leukemia cells expressing shRNA targeting FANCC (T-shFC) or a non-targeted shRNA (T-shNT) with the aldehyde 4-hydroxynonenal (4-HNE) before exposing them to the TLR-7/8 agonist R848. 4-HNE alone did not induce TNF production in either cell line but did enhance TLR-induced TNF overproduction by T-shFC cells (but not T-shNT cells), suggesting that FANCC-deficient macrophages lack factors (e.g., ALDH) that neutralize a signal-enhancing effect of this aldehyde. To directly test the effects of ALDH loss, we pretreated cells with the general ALDH inhibitor diethylaminobenzaldehyde and found that this agent enhanced R848-induced normal production of TNF by approximately 1.6-fold in control cells but did not enhance TNF production in T-shFC cells that were already overproducing TNF. Having determined that ALDH1A1 (but not ALDH2) was highly inducible in both THP-1 cells (by R848) and Lin-Sca-1+Kit+ (by TNF) murine marrow cells, we used siRNA to suppress expression of ALDH2 or ALDH1A1. Knockdown of ALDH1A1 (but not ALDH2) enhanced R848-induced normal production of TNF by approximately 1.8-fold in T-shNT but not TNF overproduction in T-shFC (fig 1) or T-shFA (FANCA knockdown) cells. Our results are consistent with the notion that ALDH1A1 is non-functional in FANCC-deficient macrophages and we confirmed that suspicion in gain-of-function analyses. Specifically, treatment of T-shNT and T-shFC cells with Alda-1 (a small molecule ALDH agonist known to enhance the activity of both ALDH1A1 and ALDH2) suppressed TLR-induced TNF production (fig 2), even in the presence of 4-HNE, by both T-shNT and T-shFC cells. In summary, (1) increasing the aldehyde load in normal macrophages has little influences on the inflammatory response induced by TLR activation but in FANCC-deficient cells aldehydes exacerbate the inflammatory response, (2) suppression of ALDH function with DEAB and specific suppression of ALDH1A1, (but not ALDH2) induces an FA-like phenotype in control macrophages, and (3) pharmacological enhancement of ALDH activity suppresses induced cytokine overproduction by FANCC-deficient macrophages. We conclude that: (1) optimal function of ALDH1A1 is FANCC-dependent in normal macrophages, (2) that the TLR-dependent overproduction of inflammatory cytokines by FANCC-deficient macrophages may result either from an increase in the aldehyde load or the loss of a non-canonical signal-suppressive function of ALDH1A1, and (3) enhancement of ALDH activity using small molecule agonists such as Alda-1 may alleviate the FA macrophage phenotype and may thereby protect HSC from inflammation-induced exhaustion. Disclosures No relevant conflicts of interest to declare.

Description: 4149 Background: The outcome of early stage breast cancer has improved in recent years with use of effective combination chemotherapy, including cyclophosphamide, anthracyclines or taxanes. Patients who undergo lumpectomy also receive radiation therapy for locoregional control. t-MDS/AML are rare but serious consequences of these therapeutic exposures; historically with an overall poor prognosis. Translocations involving the mixed-lineage leukemia (MLL) gene on chromosome band 11q23, and core binding factor genes on 21q22 and 16q22, are hallmarks of t-MDS/AML resulting from treatment with topoisomerase II inhibitors (anthracyclines). Loss of part or whole of chromosomes 5 and/or 7 (5-/7- abnormality) are associated with t-MDS/AML after treatment with alkylating agents and radiation. Allogeneic HCT (sibling-related, unrelated or double-cord) offers the only curative option for some of these patients. However, the long-term outcome of post-breast cancer t-MDS/AML after allogeneic HCT has not been described, and subgroups with better or worse prognosis not identified. Methods: From 1997 to 2011, 28 patients with t-MDS/AML after treatment of breast cancer with conventional chemotherapy and/or radiation underwent allogeneic HCT at City of Hope. Overall survival defined from the time of allogeneic HCT to the last date of follow up or date of death, was estimated using Kaplan-Meier method. Cox regression techniques were utilized to understand predictors of overall survival. Results: Of the 28 patients, 16 had been treated for early-stage breast cancer (I/IIA), 11 had received treatment for locally advanced disease (IIB/ III) and 1 patient had metastatic disease. Twenty-five patients received combination chemotherapy as adjuvant therapy or for metastatic disease; this included cyclophosphamide (100%), doxorubicin (88%) and taxanes (52%). Details of chemotherapy are unknown for one patient. Twenty-one patients (75%) also received radiation (2 received radiation alone). Median age at time of diagnosis of t-MDS/AML was 56 years (range: 34–79); median time from diagnosis of breast cancer to diagnosis of t-MDS/AML was 2.4 years (range 1–12.3); median time from t-MDS/AML to allogeneic HCT was 0.55 year (range: 0.15–4.1). Ten patients presented with MDS and 18 with AML. Cytogenetic abnormalities included MLL - 11q23 abnormality (n=12), 5-/7- and/or complex abnormalities (n=9), t(15;17)(q22;q21.1) (n=1), t(8;21) (n=1), inv(16)(p13.1;q22) (n=2). Of the 28 patients, 27 were treated with induction therapy and 74% were in complete remission (CR) at the time of HCT. Fourteen patients (50%) received sibling-related HCT, 13 received matched unrelated donor (MUD) transplant and 1 received a double cord transplant. Two patients received second allogeneic HCT and one patient received donor lymphocyte infusion (DLI) for relapsed t-MDS/AML. Sixty-one percent received reduced-intensity conditioning with fludarabine/melphalan and others received full-intensity conditioning. Sixteen patients developed cGvHD after transplant. After a median follow up of 2.2 years, 8 patients had died (cause of death: t-MDS/AML [n=5], sepsis [n=1], GvHD [n=1], breast cancer [n=1]), with an overall survival of 78.2% at two years post-HCT for the entire cohort. The 2-year overall survival was 71.4% for sibling-related HCT and 84.4% for MUD (log-rank p=0.14). There was a difference in overall survival by the type of cytogenetic abnormalities. Thus, the 2-year survival rate for patients with 11q23 abnormality was 90.9% while that for patients with chromosome 5-/7- abnormalities or complex abnormalities was 62.5% (log rank p=0.27). Patients with cGvHD had better outcome compared with those without cGvHD (2 year survival 87.5% vs. 64.3%, log-rank p value=0.03). These findings were confirmed on multivariate regression analysis, which revealed chromosome 5-/7- or complex abnormalities to be associated with a significantly worse outcome (HR=6.9, p=0.035), while cGvHD was associated with a better outcome (HR=0.14, p=0.02) after adjustment for age at HCT. Conclusions: Although t-MDS/AML after conventional treatment of breast cancer has historically been shown to have a poor prognosis, allogeneic HCT in patients with available donors provides a chance of cure. Among patients undergoing allogeneic HCT, a superior outcome was observed in patients with 11q23 abnormalities and among those with cGvHD. Disclosures: No relevant conflicts of interest to declare.

Description: Progressive bone marrow failure is a major cause of morbidity and mortality in human Fanconi Anemia patients. In an effort to develop a Fanconi Anemia murine model to study bone marrow failure, we found that Fancd2−/− mice have readily measurable hematopoietic defects. Fancd2 deficiency was associated with a significant decline in the size of the c-Kit+Sca-1+Lineage− (KSL) pool and reduced stem cell repopulation and spleen colony-forming capacity. Fancd2−/− KSL cells showed an abnormal cell cycle status and loss of quiescence. In addition, the supportive function of the marrow microenvironment was compromised in Fancd2−/− mice. Treatment with Sirt1-mimetic and the antioxidant drug, resveratrol, maintained Fancd2−/− KSL cells in quiescence, improved the marrow microenvironment, partially corrected the abnormal cell cycle status, and significantly improved the spleen colony-forming capacity of Fancd2−/− bone marrow cells. We conclude that Fancd2−/− mice have readily quantifiable hematopoietic defects, and that this model is well suited for pharmacologic screening studies.

Description: Nucleophosmin (NPM) is a multifunctional protein frequently overexpressed in actively proliferating cells including tumor and hematopoietic stem cells. Here we report that NPM protects hematopoietic cells from stress-induced apoptosis through inhibition of the tumor suppressor p53. Specifically, we forced expression of wild-type (WT) NPM or a mutant variant with a deletion of the C-terminal 120 aa of NPM (NPMΔC) by retroviral gene transfer in the granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent myeloid cell line MO7e (expresses low level of NPM) and the lymphoblast HSC536 cells derived from a Fanconi anemia (FA) patient in the C complementation group (expresses essentially undetectable NPM). Using a flow cytometric method for caspase 3 activation (early apoptosis), we demonstrated that overexpression of NPM but not the mutant NPMΔC confers MO7e and HSC536 cells resistance to apoptosis induced by growth factor deprivation and treatment with the chemotherapeutic drug etoposide. In addition, suppression of NPM expression by small interfering RNA targeting NPM in chronic myelogenous leukemia line K562 and FA-associated acute myelogenous leukemia cell line UoC-M1 increases etoposide-induced apoptosis, thus providing proof of concept evidence that the pathological elevations of NPM found in cancers and leukemias are important for maintaining cell survival and resistance to apoptosis. Because overexpression of the mutant NPMΔC, which lacks the p53-interacting domain, fails to confer cellular resistance to stress-induced apoptosis, we determined whether NPM protects cells from apoptotic cell death through a mechanism involving p53. We used the genetically matched p53 WT and null mouse bone marrow (BM) cells to show that overexpression of WT NPM protects against ionizing irradiation (IR)-induced apoptosis of wild-type but not p53-null BM cells. Moreover, NPM inhibits IR-induced p53 phosphorylation at Ser18 and transactivation, and interacts with p53 in bone marrow hematopoietic cells. Thus, this study not only demonstrates anti-apoptotic function of a proliferation-promoting protein but also suggests that cancer progression may require increased expression of NPM to suppress p53 activation and maintain cell survival.

Description: Introduction: Fanconi anemia (FA) is the most common inherited bone marrow failure syndrome. FA patients develop bone marrow failure during the first decade of life, and frequently require an allogeneic or unrelated donor bone marrow transplant. FA patients also develop other hematologic manifestations, including myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) due to clonal evolution. FA is caused by biallelic mutation in one of eighteen FANC genes, the products of which cooperate in the FA/BRCA DNA repair pathway and regulate cellular resistance to DNA cross-linking agents. Bone marrow failure in FA is attributable to an impaired hematopoietic stem and progenitor cell (HSPC) pool. HSPCs in FA patients and FA mice exhibit reduced cell number and compromised stem cell function. Recent studies suggest that bone marrow failure in FA and impaired HSPC function result from the genotoxicity of endogenous cross-linking agents or from physiological stress. A greater understanding of the mechanisms of impairment of HSPC function could improve the therapeutic options for FA patients. Using a whole genome-wide shRNA screen, we have recently identified that the canonical transforming growth factor-β (TGF-β) pathway plays an important growth suppressive role in FA and targeting this pathway can reduce the genotoxic stress-induced growth inhibition of FA cells. Here, we investigated the possible suppressive function of the TGF-β pathway in HSPCs derived from patients with FA. Methods: We performed in vitro colony-forming assays using primary FA patient- derived bone marrow CD34+ cells which were either transduced with shRNA targeting SMAD3 or treated with the anti-human TGF-β neutralizing antibody GC1008. FA-like HSPCs were generated by stably knocking down FANCD2 with lentivirus encoded shRNA in primary human cord blood CD34+ cells. An in vivo engraftment assay was performed by transplanting the FA-like HSPCs into irradiated NSG mice. Results: The primary human FA bone marrow cells displayed elevated mRNA expression of multiple TGF-β pathway components. The TGF-β pathway inhibition, by knockdown of SMAD3 or anti-human TGF-β neutralizing antibody GC1008, rescued the in vitro clonogenic defects of primary CD34+ cells from bone marrow of five different FA patients. Similarly, the TGF-β pathway disruption by depletion of SMAD3 or GC1008 antibody in primary FA-like HSPCs, also rescued their clonogenic defect, and partially restored genotoxic stress-induced growth inhibition. Further, as the very low number of CD34+ cells in FA patients did not allow efficient xenograft assay to analyze in vivo clonogenicity, we performed a surrogate in vivo xenograft assay using FA-like primary CD34+ cells. Importantly, blockade of the TGF-β pathway by GC1008 antibody treatment enhanced the engraftment potential of primary FA-like CD34+ cells in vivo. Collectively, these results demonstrated that increased TGF-β pathway signaling impairs the hematopoietic function of primary human FA HSPCs. Conclusions: The TGF-β pathway signaling is increased in primary FA patient-derived hematopoietic cells and blockade of this pathway can restore the function of human FA-deficient primary HSPCs. The TGF-β signaling pathway-mediated growth suppression may account, at least in part, for bone marrow failure in FA. This work suggests that the TGF-β signaling pathway provides a novel therapeutic target for the treatment of bone marrow failure in FA. Disclosures No relevant conflicts of interest to declare.