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Bleeding complications secondary to surgery, trauma, or coagulation disorders are important causes of morbidity and mortality. Although fibrin sealants are considered to minimize blood loss, this is not widely adopted because of its high cost and/or risk for infection. We present a novel methodology employing nonantibody fibrinogen-binding proteins, termed Affimers, to stabilize fibrin networks with the potential to control excessive bleeding. Two fibrinogen-specific Affimer proteins, F5 and G2, were identified and characterized for their effects on clot structure/fibrinolysis, using turbidimetric and permeation analyses and confocal and electron microscopy. Binding studies and molecular modeling identified interaction sites, whereas plasmin generation assays determined effects on plasminogen activation. In human plasma, F5 and G2 prolonged clot lysis time from 9.8 ± 1.1 minutes in the absence of Affimers to 172.6 ± 7.4 and more than 180 minutes (P < .0001), respectively, and from 7.6 ± 0.2 to 28.7 ± 5.8 (P < .05) and 149.3 ± 9.7 (P < .0001) minutes in clots made from purified fibrinogen. Prolongation in fibrinolysis was consistent across plasma samples from healthy control patients and individuals at high bleeding risk. F5 and G2 had a differential effect on clot structure and G2 profoundly altered fibrin fiber arrangement, whereas F5 maintained physiological clot structure. Affimer F5 reduced fibrin-dependent plasmin generation and was predicted to bind fibrinogen D fragment close to tissue plasminogen activator (tPA; residues γ312-324) and plasminogen (α148-160) binding sites, thus interfering with tPA–plasminogen interaction and representing 1 potential mechanism for modulation of fibrinolysis. Our Affimer proteins provide a novel methodology for stabilizing fibrin networks with potential future clinical implications to reduce bleeding risk.

Introduction: CPX-351 (Vyxeos ©; daunorubicin and cytarabine liposome for injection) is a dual-drug liposomal encapsulation of daunorubicin and cytarabine in a synergistic 1:5 molar drug ratio that is FDA approved for treatment of adults with newly diagnosed therapy-related AML or AML with myelodysplasia-related changes. Secondary AML is clinically and biologically similar to MDS, sharing many of the same genetic mutations. We hypothesize that CPX-351 therapy may result in deeper responses than traditional therapy with hypomethylating agents, with acceptable tolerability, and translate into better outcomes in the MDS population. This is a multicenter, dose-escalation and safety expansion study (NCT03572764) to investigate induction and consolidation therapy with CPX-351 in a transplant eligible, higher risk MDS population. Methods: Two dose levels were investigated in the dose-escalation portion. Induction Dose Level 1: (daunorubicin 29 mg/m 2 and 65 mg/m 2 cytarabine) liposome on days 1, 3, 5; and Induction Dose Level 2: (daunorubicin 44 mg/m 2 and 100 mg/m 2 cytarabine) liposome on Days 1, 3, 5. The primary objectives were to evaluate safety and to determine the dose-limiting toxicities (DLTs) and recommended Phase 2 dose (RP2D). DLTs were assessed during the induction period (up to day 56 to evaluate for prolonged myelosuppression). Additionally, a dose expansion (for up to 20 treated patients) was performed at the RP2D. After induction, patients could receive up to two cycles of consolidation per dose level: Dose Level 1, (daunorubicin 14.3 mg/m 2 and 32 mg/m 2 cytarabine) liposome on Days 1 and 3; or Dose Level 2 (daunorubicin 29 mg/m 2 and 65 mg/m 2 cytarabine) liposome on Days 1 and 3. After induction, the patient could proceed to alloHCT at any time at the discretion of the treating physician. Key eligibility criteria included MDS patients who were naïve to hypomethylating agents, aged 18-70 years, an IPSS-R score > 3 (Intermediate, High or Very High Risk), ≥ 5% bone marrow myeloblasts, and suitable candidates for cytotoxic induction therapy and allogeneic hematopoietic cell transplant (alloHCT). Key secondary endpoints were day 30 and 60 post-induction mortality, the proportion of patients proceeding to alloHCT, and response assessments per IWG 2006 criteria. Results: As of abstract submission, 19 patients have been treated and dose escalation is complete. The dose escalation portion included 12 patients (Dose Level 1, n=6; Dose Level 2, n=6), and the safety expansion to date includes 7 patients treated at Dose Level 2. The median age was 64 years (range, 18-68), 67% were female, and the IPSS-R risk categories were as follows: intermediate (n=1, 5%), high (n=10, 53%) and very high (n=8, 42%). There were no DLTs in either Dose Level 1 or Dose Level 2, and Dose Level 2 was selected for safety expansion. Treatment-emergent adverse events (TEAEs) were evaluated in the 18 patients who had completed induction (Table). The most common TEAEs were hematologic, as expected. The most common non-hematologic TEAEs were febrile neutropenia (n=13, 72.2%), hypertension (n=9, 50%), and sepsis (n=5, 27.8%). SAEs included febrile neutropenia (n=5), sepsis (n=2), lower GI hemorrhage (n=2, two instances in the same patient), atrial fibrillation (n=1), pneumonitis (n=1), and catheter related infection (n=1). To date, the 30 and 60 day mortality is 0% and 5% (n=1), respectively, with the 1 death unrelated to therapy and due to progression of disease to AML. Of the 19 patients, 13 have received alloHCT with 4 still potentially alloHCT candidates. Of response evaluable pts (n=18, 1 pt pending response evaluation at data cutoff), the overall response rate was 78% with best overall responses of CR (n=4), mCR (n=10), stable disease (n=2), progressive disease (n=2) and pending (n=1). Of the 10 patients with mCR, 3 also had hematologic improvements. Conclusions: CPX-351 (daunorubicin 44 mg/m 2 and 100 mg/m 2 cytarabine) liposome on Days 1, 3, 5, has demonstrated a tolerable safety and promising efficacy profile when used in a transplant eligible, higher risk MDS population, warranting further study. Updated safety and efficacy outcomes will be presented at the meeting. Figure 1 Figure 1. Disclosures Jacoby: Abbvie: Research Funding; Jazz: Research Funding. Sallman: Shattuck Labs: Membership on an entity's Board of Directors or advisory committees; Kite: Membership on an entity's Board of Directors or advisory committees; Intellia: Membership on an entity's Board of Directors or advisory committees; Magenta: Consultancy; Incyte: Speakers Bureau; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Syndax: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees; Aprea: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Consultancy; AbbVie: Membership on an entity's Board of Directors or advisory committees. Scott: Bristol Myers Squibb: Consultancy, Honoraria, Research Funding. Komrokji: AbbVie: Consultancy; Acceleron: Consultancy; Geron: Consultancy; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; PharmaEssentia: Membership on an entity's Board of Directors or advisory committees; Jazz: Consultancy, Speakers Bureau; Taiho Oncology: Membership on an entity's Board of Directors or advisory committees; BMSCelgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Uy: AbbVie: Consultancy; Macrogenics: Research Funding; Agios: Consultancy; GlaxoSmithKline: Consultancy; Novartis: Consultancy; Genentech: Consultancy; Astellas: Honoraria, Speakers Bureau; Jazz: Consultancy. OffLabel Disclosure: CPX-351 (Vyxeos©; daunorubicin and cytarabine liposome for injection) is FDA approved for treatment of adults with newly diagnosed therapy-related AML or AML with myelodysplasia-related changes

Aggressive non-Hodgkin's lymphomas (NHLs) [diffuse large b-cell lymphoma (DLBCL), grade 3b follicular lymphoma, and mantle cell lymphoma (MCL)] have dismal cure rates, especially in the relapsed/refractory setting, substantiating the need for novel treatment strategies to improve outcomes. Here we show that p21-activated kinase 4 (PAK4) and nicotinamide phosphoribosyltransferase (NAMPT) are essential for lymphoma subsistence and dual-targeting PAK4-NAMPT by the small molecule inhibitor KPT-9274 (orally bioavailable phase 1 compound), previously shown to be cancer cell-selective, acts by energy depletion, inhibiting cell proliferation and eventual apoptosis as a reasonable treatment strategy for aggressive NHLs. To demonstrate PAK4-NAMPT is essential for lymphoma survival, we showed increased PAK4 expression in primary NHL tissue (n=94) than in normal lymph nodes (n=8) (Fig1A). Lymphoma cell lines were sensitive to the PAK4/NAMPT dual inhibitor KPT-9274 (induced apoptosis with an EC 50 of 95.17 nM in WSU-DLCL2 and 13.9 nM in WSU-FSCCL cells). Similar results were obtained with positive controls PAK4 [PF-3578309] and NAMPT specific inhibitors [FK-866]. RT-PCR confirmed PAK4 and NAMPT down-expression in KPT-9274 treated WSU-FSCCL cells. Western blot of WSU-DLCL2 and WSU-FSCCL cells treated with KPT-9274 showed significant reductions in BCL-2 and enhanced cleaved-Caspase-3 and cleaved-PARP expression. We then assessed the impact of KPT-9274 on the cellular ATP and NAD pool since NAMPT inhibition can suppress both and induce cell death. NAD/NADH and ATP levels decreased in both cell lines [WSU-DLCL2 & WSU-FSCCL], with increasing KPT-9274 concentrations (Fig.1B&1C). We then showed KPT-9274 to synergize with the standard of care chemotherapy cyclophosphamide, vincristine, and adriamycin used for NHL management (decreased IC25 and IC50 in several combination treatments). In lymphoma xenografts, mice with transplanted WSU-DLCL2 fragments showed ~50% reduction in tumor volume (p 150-day increase in host life span compared to control and 3/6 mice were cured) (Fig.1F). After day 150, the remaining mice were dissected to confirm cures. Blood smears drawn from mice post three weeks of KPT-9274 treatment showed a significant reduction of circulating WSU-FSCCL cells validating its efficacy systemically (Fig.1G). Since exogenous niacin can regenerate NAD regulated by NAMPT in a rate-limiting step, the impact of niacin co-dosing with KPT-9274 was evaluated on tumor growth using the Z-138 MCL tumor xenograft model in nude mice. Tumor-bearing mice were treated with vehicle, KPT-9274 (200 mg/kg BID), niacin (30 mg/kg), or a combination of KPT-9274/niacin. All groups that received KPT-9274 treatment showed reductions in tumor size, while the vehicle control and niacin alone groups showed a rapid increase in tumor volume. Both KPT-9274 and combo groups showed a significant decrease in tumor growth compared to control (p=0.0002); however, there were no statistically significant differences between the KPT-9274 and the KPT-9274/niacin combo (Fig.1H). Similarly, in another MCL xenograft model, KPT-9274 resulted in a statistically significant reduction in JeKo-1 tumors compared to control (p = 0.0059). Niacin did not affect the tumor growth suppression induced by KPT-9274 treatment. No animals died in this study. Thus, supplementing niacin with KPT-9274 may alleviate the adverse effect without compromising the efficacy of the treatment. Also, KPT-9274 treated Z-138 residual tumors showed a reduction in PAK4, GEF-H1, paxillin, vinculin, cyclin D1, Dvl2, and Ki67 and enhancement in apoptosis by IHC staining (Fig.1I). The anti-tumor potential of KPT-9274 ± niacin in several aggressive lymphoma models strongly supports its efficacy in this setting. It strengthens our phase I study design to evaluate safety and tolerability and anti-tumor activity in patients with advanced solid malignancies or NHL (NCT02702492). Figure 1 Figure 1. Disclosures Balasubramanian: Servier Pharmaceuticals: Research Funding. Zonder: Caelum Biosciences: Consultancy; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Research Funding; Amgen: Consultancy; Alnylam: Consultancy; Regeneron: Consultancy; Janssen: Consultancy; Intellia: Consultancy. Senapedis: Karyopharm Therapeutics: Ended employment in the past 24 months, Patents & Royalties; Omega Therapeutics: Current Employment; Restorbio: Other: Personal fees. Baloglu: Karyopharm Therapeutics: Current Employment, Other: Personal fees. Shah: Karyopharm Therapeutics Inc.: Current Employment, Current equity holder in publicly-traded company. Landesman: Karyopharm Therapeutics: Current Employment, Current equity holder in publicly-traded company. Shacham: Karyopharm: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties: (8999996, 9079865, 9714226, PCT/US12/048319, and I574957) on hydrazide containing nuclear transport modulators and uses, and pending patents PCT/US12/048319, 499/2012, PI20102724, and 2012000928) . Kauffman: Karyopharm Therapeutics Inc.: Current Employment, Current equity holder in publicly-traded company. Azmi: Karyopharm Therapeutics: Research Funding.

Introduction: Haploidentical hematopoietic cell transplantation (haplo-HCT) is an increasingly utilized therapy for a variety of hematologic malignancies. Determining which donor characteristics affect transplant outcomes is of particular interest in haplo-HCT, as there are often multiple donors available for a given patient. A survival benefit with younger donors has been reported in some recent observational studies (DeZern et. al., Blood Advances, March 2021); (Canaani et. al., AJH, Sep. 2017). A decrease in non-relapse mortality (NRM) and increase in relapse with no overall survival difference associated with younger donors has also been observed (Mariotti et. al., Blood Advances, June 2020). These previous studies have utilized populations with bone marrow as the predominant stem cell source. Solomon et al. (BBMT Sep. 2018) observed poorer survival, increased relapse, and worse NRM with parent donors relative to children in a largely peripheral blood population. HLA DR and DP mismatch were noted to be associated with improved survival. Here we describe outcomes in peripheral blood haplo-HCT and their association with potentially selectable donor characteristics including age and relationship to the patient. Patients and Methods: We performed a retrospective review of patients who underwent peripheral blood haplo-HCT with PtCy from July 2009 through May 2021. A total of 323 patients were identified with AML (205), ALL (43), MDS (26), and other (49). Univariate and multivariate analyses (MVA) were conducted examining the effect of donor characteristics on overall survival (OS), NRM, relapse, acute and chronic GVHD. Donor characteristics included age, relationship, ABO status, CMV status, and HLA match grade. We controlled for patient characteristics known to affect outcomes including disease type, DRI, HCT CI, KPS, active disease at transplant, myeloablative conditioning, and prior HCT. Results: Median donor age was 40 (range 15-71) with male predominance (64%). Most were ABO compatible (63%) - 12% had major ABO mismatch, 20% minor, and 4% bidirectional. Donor-recipient CMV status matched in 61% of pairs, 13% were donor positive-recipient negative, 26% donor negative-recipient positive. Most were 5/10 HLA matched (51%) with 20% 6/10 and 13% 7-9/10. Univariate analysis revealed that increasing donor age was associated with higher NRM (HR 2.29, p=0.005 for donors age 30-44; HR 2.06, p=0.012 age > 44) but lower relapse risk (HR 0.56, p=0.012 age 30-44; HR 0.69, p=0.10 age > 44). There were no differences in aGVHD or cGVHD based on donor characteristics in univariate analysis. In MVA, relapse risk was lower in patients with older donors , p=0.046). In contrast, NRM was higher in patients with older donors (HR 1.73 age 30-44, HR 1.69 age > 44, p=0.010). There was no difference in overall survival based on donor age (HR 1.23 age 30-44, HR 1.38 age > 44, p=0.11). We next examined the effect of donor relationship on outcomes while controlling for donor age, patient age, and patient disease risk factors. We found no difference in outcomes between parent, sibling, or child donors. Conclusions: Increasing donor age was associated with lower relapse risk but higher NRM. These competing effects resulted in no difference in OS based on donor age. Other donor factors including relationship (parent / sibling / child), CMV status, ABO mismatch, donor sex, and HLA match grade were not associated with outcomes. Solomon et al. reported better outcomes with child compared to parent donors, a finding not replicated here, however our analysis controlled for donor age which could have been a proxy for relationship in their study. These data suggest that in peripheral blood haplo-HCT, younger donors may be preferred in patients with high risk of transplant related complications. In contrast, older donors may be preferred in patients where relapse risk is high. Data on HLA-DR and DP match is being analyzed and will be presented at the ASH 2021 meeting. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Introduction: Cytokine release syndrome (CRS) is a potentially fatal systemic inflammatory response that can occur in patients undergoing haploidentical hematopoietic cell transplantation (haplo-HCT). IL-6 inhibitors, such as tocilizumab, are effective therapy in moderate to severe cases. Several studies have shown there to be a significant increase in infections with the use of tocilizumab in patients with rheumatoid arthritis, where it is given on a long term basis unlike in the post-haplo-HCT setting, where tocilizumab is rarely given for more than a few days. Severe CRS has been associated with increased infection risk in prior studies. However, the effect of anti-IL-6 therapy on infection risk has not been well established in the early haplo-HCT setting. In this study, we examined the effect of tocilizumab for treatment of CRS on the incidence of blood stream infections (BSIs) in the early post peripheral blood haplo-HCT setting. Patients and Methods: We performed a retrospective analysis of 235 patients who underwent T cell-replete peripheral blood haplo-HCTs from 2013 to 2020, stratified on CRS grade (graded by Lee criteria) and tocilizumab administration, for incidence of BSI. Positive blood cultures during days +2 to +28 post-haplo-HCT were included. Patients with positive blood cultures during the immediate peri-transplant period (days -5 to +1) were excluded as infection preceded CRS and tocilizumab administration. Patients who had positive blood cultures within 48 hours of CRS diagnosis were excluded as sepsis cannot be distinguished from CRS. Results: The overall incidence of bloodstream infection was 17% with 41 out of the total 235 patients having positive blood cultures. Patients with mild CRS had lower incidence of infection than patients with severe CRS (OR 0.31, 95% CI 0.13-0.74, p=0.0086). In the tocilizumab group, 31% (15/49) of patients had positive blood cultures compared with 14% (26/186) in the non-tocilizumab group (OR 1.61, 95% CI 0.30-8.60, p=0.58). However, after controlling for CRS grade, tocilizumab administration was not associated with higher rates of BSIs for any grade of CRS. Conclusions: Severe CRS after haplo-HCT is associated with higher risk of early BSI. However, tocilizumab therapy does not further increase risk of BSI in the early post-haplo-HCT setting. These data suggest that tocilizumab, a potentially life saving therapy, can be given without increasing risk of blood stream infections after haplo-HCT. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Acute leukemias (AL) is a major cause of cancer death in young age. Extensive research is being conducted to identify novel and innovative approaches for leukemia treatment. Transcriptomic and epigenetic studies might help to discover potential targets, paving the way for molecular-targeted therapies. We analyzed a cohort of 34 AL at diagnosis: 10 pediatric Acute Lymphoblastic Leukemias (8 B-cell, 1 T-cell and 1 bi-phenotypic ALL), 4 young adult ALL (2 B-cell and 2 T-cell phenotype), and 20 pediatric Acute Myeloid Leukemias (AML: 3 M1, 4 M2, 1 M3, 5 M4, 7 M5) with an average blasts population higher than 80% (85+/-11% in ALL; 83+/-13% in AML). Six out of 14 ALL, and 12 out of 20 AML fell under "high risk" category according to clinical standard risk stratification algorithms. On all patients we performed mRNA sequencing (20-million-reads on Illumina Hiseq 4000). Analyses were performed adjusting The expression of a set of 800 microRNAs (miRNAs) was evaluated by means of Nanostring miRNA panel. Expression signatures and associations among the different risk groups were calculated with t-tests and linear regression analyses. Applying stringent FDR statistical measurement, we discovered 3 genes that significantly differentiate the transcriptomic profile of high vsintermediate/standard-risk ALL in mRNAseq. The expression of PGR3 (p53 Responsive Gene) and long-non-coding RNAs (lncRNAs) ENSG00000228737 and ENSG00000253174 were respectively 45.5, 4.2 and 3.9 time downregulated in high-risk ALL. To explore more deeply the apoptosis pathway in ALL, we measured Tp53 expression and found it significantly downregulated in the high-risk vsintermediate-standard-risk ALL (p= Tp53 dysregulation is a known hallmark for tumor progression; Tp53 mutations - ranging from 1-2% to 10% in pediatric and adult ALs - correlate with worse prognosis. However, in our cohorts, this gene signature was found significant only in high-risk ALL, homogeneously distinguishing them from intermediate/standard-risk ALL. Transcriptome clinical variant analyses excluded pathogenetic known variants that could explain such marked difference. Also, it is unlikely that somatic genetic mutations acquired by the tumor would explain such a homogeneous behavior of high-risk ALL. Thus, we analyzed the p53 regulatory pathway. Interestingly we found that miRNAs known to be involved in p53 control were significantly upregulated in high-risk vs intermediate-standard-risk ALL (p We found PRG3 and Tp53 significantly downregulated in high-risk ALL, with PRG3 expression 45 times lower than intermediate/low-risks ALL. Deeper analyses pointed out to an apoptosis control program not generated by a somatic mutation in the tumor, nor a germline clinical patient variant, but by an epigenetic mechanism. We are currently validating these data in a larger cohort, adding also methylome analyses. It will be interesting also to explore the function of the lncRNAs markedly downregulated in our cohort, whose functions are still unknown or partially known. Because of the small numerosity of the ALL high-risk cohort, we were not able to dissect high-risk young adults (4/6) from pediatric ALL (2/6). Although the homogeneity of data suggests a shared apoptosis control mechanism, it will be worthy to explore in a larger cohort whether the general worse prognosis of young adults/adults vs pediatric ALL is at least partially explained by this mechanism. Disclosures No relevant conflicts of interest to declare.

Background: Ibrutinib (I) and venetoclax (V) have each demonstrated modest single-agent activity in relapsed/refractory follicular lymphoma (FL) (Gopal A, JCO 2018; Davids M, JCO 2017). Preclinical data have shown synergy with these agents in B-cell cell lines (Kuo H, Mol Cancer Ther 2017). Based on these observations, we proposed the first trial to combine I and V in FL. Results from the phase Ib portion of this multi-institutional investigator-initiated trial are presented here (NCT02956382). Methods: This phase Ib/II trial is open at Georgetown/Lombardi CCC, Hackensack/John Theurer CC, and University of Washington/Fred Hutchinson/Seattle Cancer Care Alliance. Eligibility criteria include WHO grade 1-3a FL, >1 prior systemic therapy, measurable disease warranting therapy by standard criteria or physician discretion, ECOG performance status < 2, adequate marrow, hepatic, renal function. Patients (pts) were enrolled in a standard phase I 3+3 design at a starting dose level (DL) of I 420 mg daily, V 400 mg daily (DL0). The highest initially planned dose level was DL3: I 560 mg daily, V 800 mg daily. There was no dose ramp up of V based on monotherapy experience in FL. Pts at high risk for tumor lysis syndrome (TLS), defined as node ≥ 8 cm and/or significant lymphocytosis, were hospitalized for initial dose. Pts received study drugs until progression or unacceptable toxicity. Response was assessed by PET-CT and bone marrow biopsy (if marrow involvement present at time of enrollment). Results: Sixteen pts were enrolled between November 2017 - May 2020. Median age was 66 years (range 50-87); 75% were male; 75% were Stage III/IV, 94% had WHO grade 1/2 FL (Table 1). FLIPI score at enrollment was 25% low risk, 44% intermediate risk, 31% high risk. Two pts were considered high risk for TLS. Pts received a median of 2 prior therapies (range 1-8); 19% were refractory to last line of therapy. Cohort enrollment was: DL0 (n=3), DL1 (n=6), DL2 (n=6), DL3 (n=1). The protocol was amended to close DL3 based on pharmacokinetic data from DL2 indicating a 1.8-fold higher mean steady-state ibrutinib plasma exposure compared to ibrutinib 560 mg monotherapy and concern for potential toxicity. Grade 3 adverse events (AE) included neutropenia (25%), thrombocytopenia (13%), lung infection (13%), upper respiratory infection (6%), neutropenic fever (6%), atrial fibrillation (6%), ALT/AST elevations (6%), mucositis (6%), failure to thrive in setting of progression (6%), abdominal pain (6%). There were no grade 4/5 AE. Grade 1/2 AE occurring in > 20% of pts included diarrhea (75%), nausea (63%), bruising (38%), rash (31%), headache (31%), constipation (25%), fatigue (25%). There was no evidence of clinical TLS; 19% had grade 1 hyperuricemia. The pt enrolled at DL3 had grade 1 diarrhea, grade 1 neutropenia. One dose limiting toxicity (DLT) occurred at DL1 (I 560 mg, V 400 mg): grade 3 neutropenia with fever and infection. There were no other DLTs. Therefore, DL2 (I 560 mg, V 600 mg) was determined to be the recommended phase 2 dose (RP2D). The ORR was 69% (0.413, 0.890); CR 25% (0.073, 0.524). The ORR at the RP2D was 83% (CR 33%). Responses by dose level are listed in Table 2. The regimen demonstrated activity in the bone marrow; 2 pts had eradication of involvement and 1 had a decrease from 60% to 0.5% by flow cytometry. Response by lines of prior therapy: 1 (86%, 6/7), > 2 (56%, 5/9). Most pts (91%) had a response by time of first assessment (12 weeks). The median progression-free survival (PFS) was 8.3 months (5.6 months, NA) (Figure 1). Of note, 2 responding pts chose to withdraw from study due to travel and were censored in the PFS analysis at time of discontinuation. One remained in a CR at least 9 months after study withdrawal as documented by PET-CT performed off protocol. No pts discontinued due to toxicity. Conclusion: In the first clinical trial to combine a BTK inhibitor and a BCL-2 inhibitor in relapsed/refractory FL, we found the I-V doublet to demonstrate a toxicity profile similar to that seen in mantle cell lymphoma and CLL. While our sample size is small, there was no evidence of clinical TLS, despite omission of the V ramp up. Preliminary results of anti-tumor activity are encouraging and further evaluation at the RP2D (I 560 mg, V 600 mg) is ongoing in the phase II trial. The combination of ibrutinib and venetoclax may provide an effective option for FL, utilizing a targeted approach distinct from other novel agents currently approved for this malignancy. Disclosures Ujjani: Verastem Oncology: Consultancy, Honoraria; Genentech: Consultancy, Honoraria; MorphoSys: Consultancy; Atara: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria, Research Funding; Gilead/Kite: Consultancy, Research Funding. Lai:Agios: Consultancy; Macrogenics: Consultancy; Astellas: Speakers Bureau; Jazz: Speakers Bureau; Abbvie: Consultancy. Leslie:Seattle Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; KitePharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Speakers Bureau; Celgene: Speakers Bureau; BeiGene: Honoraria, Speakers Bureau; Pharmacyclics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AstraZeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Epizyme: Honoraria, Speakers Bureau; Karyopharm: Honoraria, Speakers Bureau; Bayer: Consultancy, Membership on an entity's Board of Directors or advisory committees; ADC therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: 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. Broome:sanofi: Honoraria; argenx: Honoraria; apellis: Honoraria; Alexion: Honoraria. Gopal:IgM bio, BMS, merck: Research Funding; imab bio, takeda,astrazeneca,gilead: Research Funding; Seattle Genetics; Janssen; IMab Bio; TG Therapeutics; Astra Zeneca; Merck; Gilead; ADC Therapeutics; Nurix; TG therapeutics, Cellectar; Actinium: Consultancy; Seattle Genetics; Janssen; Takeda; IgM Bio; IMab Bio; BMS; Astra Zeneca; Merck; Gilead: Research Funding. Smith:Beigene: Consultancy; Millenium/Takeda: Consultancy; AstraZeneca: Consultancy; Portola: Research Funding; Seattle Genetics: Research Funding; Pharmacyclics: Research Funding; Merck: Research Funding; Incyte: Research Funding; Ignyta: Research Funding; Genentech: Research Funding; De Novo Biopharma: Research Funding; Bristol Meyers Squibb: Research Funding; Ayala: Research Funding; Acerta Pharma BV: Research Funding; AstraZeneca: Research Funding; Bayer: Research Funding; Karyopharm: Consultancy. Till:Mustang: Patents & Royalties, Research Funding. Lynch:Morphosys: Consultancy; Takeda: Research Funding; Bayer: Research Funding; TG therapeutics: Research Funding; Incyte: Research Funding; Juno: Research Funding; Cyteir: Research Funding; Genentech: Research Funding; Rhizen: Research Funding. Shadman:Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; TG therapeutics: Research Funding; Celgene: Research Funding; Sunesis: Research Funding; Gilead: Research Funding; AstraZeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Meyers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees; Verastem: Consultancy, Membership on an entity's Board of Directors or advisory committees; ADC Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Atara Biotherapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cellectar: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mustang Bio: Research Funding; BeiGene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta Pharma: Ended employment in the past 24 months; MophoSys: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sound Biologics: Consultancy, Membership on an entity's Board of Directors or advisory committees. Maloney:Novartis: Consultancy, Honoraria; Bioline Rx: Consultancy, Honoraria; Juno Therapeutics: Consultancy, Honoraria, Patents & Royalties: Patents are pending, but not issued, licensed, no royalties, no licensees., Research Funding; A2 Biotherapeutics: Consultancy, Current equity holder in publicly-traded company, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Kite, a Gilead Company: Consultancy, Honoraria, Research Funding; Gilead Sciences: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; MorphoSys: Consultancy, Honoraria; Genentech: Consultancy, Honoraria; Pharmacyclics: Consultancy, Honoraria. Cheson:TG Therapeutics: Speakers Bureau; Symbio: Membership on an entity's Board of Directors or advisory committees; Kite: Consultancy; Karyopharm: Consultancy, Membership on an entity's Board of Directors or advisory committees; Morphosys: Consultancy; Trillium: Research Funding; Abbvie: Consultancy, Research Funding; Jannsen: Consultancy; Pharmacyclics: Consultancy, Research Funding; Parexel: Consultancy; GSK: Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: We are presenting data regarding the use of venetoclax and ibrutinib in follicular lymphoma.

Introduction: Acute GVHD (aGVHD) contributes to poor outcomes and increased healthcare resource utilization (HRU) after allogeneic stem cell transplantation (allo-HCT). However, HRU and the economic burden of aGVHD based on severity of the disease and organ involvement is not well characterized. We examined the HRU, cost and mortality associated with aGVHD severity from initial hospitalization (index admission) up to 100 days post allo-HCT. Methods: Study cohort included 290 adult (≥ age 18) recipients of a first allo-HCT at the University of Florida between 1/2010 and 1/2019. The electronic medical records were reviewed for all patients who developed aGVHD as well as 116 patients without aGVHD who lived at least 1 month after HCT. Clinical measures that characterize the severity of aGVHD and extent of organ involvement were collected from electronic medical records. Medical costs and total hospital days were retrieved from administrative data that allocate costs to services based on departmental input for resource use and were adjusted to 2018 dollars. Wilcoxon rank sum test was used to compare number of inpatient days and total cost. Chi-squared test was used to compare ICU admission rate. Multivariable linear regression was fitted on log transformed cost. Results are shown as cost multipliers that represent ratios on original cost scale. Results: Of the 290 patients, 174 developed aGVHD within 100 days of allo-HCT. A higher proportion of patients with aGVHD had a Karnofsky performance status Development of aGVHD was associated with a significantly higher total (inpatient and outpatient) cost. The mean total cost for patients with and without aGVHD were $226,545 and $165,622, respectively (P Conclusion: HRU, cost, and clinical outcomes were associated with the severity of aGVHD. Development of higher grades of aGVHD and LGI aGVHD were associated with a poor clinical outcome and considerably increased healthcare economic burden. Given these clinical and economic risks it is imperative that new therapeutic strategies are developed for this patient population. Disclosures Farhadfar: Incyte pharmaceutical: Other: Member of GVHD advisory forum; CSL Behring: Research Funding. Leather:CSL Behring: Research Funding. Itzler:CSL Behring: Current Employment, Current equity holder in private company. Wingard:CSL Behring: Research Funding.

Introduction: The chemoimmunotherapeutic regimen of rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) is curative for many patients (pts) with diffuse large B cell lymphoma (DLBCL), but a subset of high-risk pts, including those with transformed and double-hit lymphomas, are less likely to be cured by R-CHOP. Exportin-1 (XPO1), a nuclear export protein, is overexpressed in NHL and correlates with chemotherapy-resistance and poor prognosis. Overexpression of XPO1 results in increased nuclear export of tumor suppressor proteins (TSPs) leading to their functional inactivation, and enhanced expression of Bcl-2 family members and c-Myc. Inhibition of XPO1 leads to TSP activation and oncoprotein reduction. Selinexor, a novel, oral selective inhibitor of XPO1, was approved by the FDA for DLBCL after 2 prior therapies based on an overall response rate (ORR) of 29% including a 13% complete response (CR) rate. Our previous preclinical results in vitro and in vivo showed potent additive or synergistic cytotoxicity of selinexor with chemotherapy in xenograft NHL mouse models. We evaluated the safety and preliminary efficacy of the combination of selinexor with R-CHOP chemotherapy in pts with untreated NHL. Methods: This is an open-label phase 1b study to determine the safety, tolerability, recommended phase 2 dose (RP2D) and maximum tolerated dose (MTD) of selinexor in combination with R-CHOP in pts with NHL. Pts with stage III/IV DLBCL (including transformed DLBCL) who had no prior therapy, and pts with indolent lymphomas who had either no prior therapy or received one prior therapy not containing an anthracycline, were included. A standard 3 + 3 dose escalation design in which pts received 6 cycles of R-CHOP (rituximab 375 mg/m2, cyclophosphamide 750 mg/m2, doxorubicin 50 mg/m2 IV, vincristine 0.5 mg/kg on day 1, prednisone 100 mg po on days 1-5 in a 21 day cycle) and 60 mg or 80 mg selinexor on days 1, 8, and 15 of each cycle was used. Weekly selinexor maintenance, at the dose employed at the conclusion of R-CHOP, was continued for an additional year. Blood samples were obtained on days 0, 3, 5, 8 and 15 for RNA isolation followed by RT-PCR to evaluate the expression of targets or downstream effectors including XPO1, AABCG2, AKT, Bcl-2, Bax and ERK. Results: A total of 12 pts were enrolled: 10 pts had DLBCL (1 GCB, 5 non-GCB, 4 transformed from indolent NHL); 2 pts had follicular lymphoma. Median age was 55 years. Six pts received selinexor 60 mg weekly + R-CHOP and 6 pts received selinexor 80 mg weekly + R-CHOP. Among the first 3 pts in the 60 mg cohort, there was an episode of grade 3 supraventricular tachycardia and an episode of grade 3 syncope. No further cardiac events occurred amongst these 3 pts, and this cohort was expanded by 3 pts. No further serious cardiac adverse events (AEs) occurred for the entire cohort. Among the first 3 pts in the 80 mg cohort, 1 DLT occurred: grade 3 nausea and vomiting. There were more discontinuations and dose reductions in the 80 mg cohort than in the 60 mg cohort; The majority of AEs were grade 1 or 2 and included nausea (100%), fatigue (67%), skin and nail changes (58%), constipation (42%), dizziness (42%), sinus congestion (42%), and vomiting (42%) with nausea and fatigue lasting ~1-2 days. AEs leading to selinexor discontinuation in 5 pts included fatigue +/- nausea. Dose reductions occurred in 3 pts, all due to fatigue. 8 pts completed all 6 cycles of R-CHOP with Selinexor, and 6 continued maintenance therapy. 2 pts came off study for non-compliance. MTD was not reached, however because of the higher frequency of selinexor discontinuation and dose reductions in the 80 mg cohort, selinexor 60 mg weekly + R-CHOP is the recommended RP2D. Among 10 efficacy evaluable pts (6 at 60 mg and 4 at 80 mg), the ORR was 100%: 9 pts with a CR (including 4 non-GCB pts) and 1 with a PR. Median follow up is 476 days. In line with previous reports, XPO1 mRNA expression was consistiently increased in all pt blood samples at day 3. Conclusions: R-CHOP in combination with weekly selinexor showed encouraging preliminary efficacy with an ORR of 100% and CR of 90%. At the 60 mg selinexor dose level, AEs were manageable and milder (grade 1/2) than those previously reported with selinexor. The results of correlative studies performed were consistent with our preclinical findings. A phase 2 study of this combination is ongoing in frontline DLBCL and Richter's transformation (NCT03147885). Disclosures Seymour: Incyte: Research Funding; Janssen/Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Research Funding; Genentech: Research Funding; Seattle Genetics: Research Funding; Bristol-Myers Squibb: Research Funding. Ramchandren:Seattle Genetics, Sandoz-Novartis, Pharmacyclics, an AbbVie Company, Janssen, Bristol-Myers Squibb: Consultancy; Merck, Seattle Genetics, Janssen, Genentech: Research Funding. Yang:Protagonist: Research Funding; AROG: Research Funding; AstraZeneca: Research Funding; Jannsen: Research Funding. Bhutani:Sanofi: Consultancy, Research Funding. Azmi:Rhizen Phamaceuticals Inc, EISAI: Research Funding; GLC, Guidepoint: Consultancy. Zonder:Intellia, Amgen, Takeda, Janssen, Regeneron, Alnylam, Caelum, Oncopeptides: Consultancy; BMS, Celgene: Research Funding.

The use of post-transplant cyclophosphamide (PTCy) in the context of haploidentical stem cell transplant (haplo-SCT) has led to drastically reduced rates of Graft-vs-Host (GvH) disease through selective depletion of highly allo-reactive donor T-cells. Early trials utilized a reduced-intensity Flu/Cy/TBI preparative regimen and bone marrow grafts; however, relapse rates remained relatively high (Luznik et al. BBMT. 2008). This led to the increased use of myeloablative (MA) regimens for haplo-SCT, which have been associated with decreased relapse rates (Bashey et al. J Clin Oncol. 2013). Most studies have used a MA total body irradiation (TBI) based regimen for haplo-SCT. Preparative regimens using fludarabine and melphalan (FluMel), with or without thiotepa, ATG, and/or low dose TBI have also been reported using bone marrow grafts. Reports on the safety and toxicity of FluMel in the haplo-SCT setting with PTCy and peripheral blood stem cell (PBSC) grafts are lacking. In this two-center retrospective analysis, the safety/toxicity of FluMel as conditioning for haplo-SCT was evaluated. We report increased early mortality and toxicity using standard FluMel conditioning and PBSC grafts for patients undergoing haplo-SCT with PTCy. 38 patients at the University of Rochester Medical Center and the Washington University School of Medicine underwent haplo-SCT with FluMel conditioning and PBSC grafts between 2015-2019. Outcomes were measured by retrospective chart review through July 2019. 34 patients (89.5%) received FluMel(140 mg/m2). Two patients received FluMel(100 mg/m2) and two patients received FluMel(140 mg/m2) + ATG. The median age at time of haplo-SCT was 60 years (range 21-73). 20 patients were transplanted for AML, eight for MDS, two for PMF, two for NHL, and five for other malignancies. The median Hematopoietic Cell Transplantation-specific Comorbidity Index (HCT-CI) score was 4 (≥3 indicates high risk). 11 patients had a history of prior stem cell transplant, and 16 patients had active disease prior to their haplo-SCT. Seven patients had sex mismatch with their stem cell donor. Median donor age was 42 (range 21-71). 20 patient deaths occurred by July 2019 with a median follow up of 244 days for surviving patients. Nine patients died before day +100 (D100, "early mortality"), with a D100 non-relapse mortality (NRM) rate of 24%. Median overall and relapse free survival (OS and RFS, respectively) were 197 days (95% CI 142-not reached) and 180 days (95% CI 141-not reached), respectively, for the entire cohort. The 1 year OS and NRM were 29% and 50%. The incidence of grades 2-4cytokine release syndrome (CRS) was 66%, and 52% of these patients were treated with tocilizumab. CRS was strongly associated with early mortality, with D100 NRM of 36% in patients with grade 2-4 CRS compared to 0% in those with grade 0-1. The incidence of acute kidney injury (AKI) was 64% in patients with grade 2-4 CRS, and 8% in those without (p < 0.001). 28% of patients with AKI required dialysis. Grade 2-4 CRS was seen in 54% of patients in remission prior to haplo-SCT and in 92% of those with active disease (p = 0.02). Of the 9 patients with early mortality, 89% had AKI, 44% needed dialysis, and 100% had grade 2-4 CRS, compared to 31%, 10%, and 55% in those without early mortality (p = 0.002, p = 0.02, p = 0.01). Early mortality was not significantly associated with age, HCT-CI score, second transplant, disease status at transplant, total dose of melphalan, volume overload/diuretic use, or post-transplant infection. In conclusion, we observed a very high rate of NRM with FluMel conditioning and PBSC grafts for haplo-SCT with PTCy. The pattern of toxicity was strongly associated with grade 2-4 CRS, AKI, and need for dialysis. These complications may be mediated by excessive inflammation in the context of allo-reactive donor T-cell over-activation. Consistent with this, multiple groups have shown that FluMel conditioning in haplo-SCT is safe when using bone marrow or T-cell depleted grafts. Based on our institutional experiences, we would discourage the use of FluMel as conditioning for haplo-SCT with PTCy with T-cell replete PBSC grafts. Alternative regimens or variations on melphalan-based regimens, such as fractionated melphalan dosing or inclusion of TBI may improve outcomes but further study and randomized controlled trials are needed. This study is limited in its retrospective design and sample size. Figure Disclosures DiPersio: WUGEN: Equity Ownership, Patents & Royalties, Research Funding; Karyopharm Therapeutics: Consultancy; Magenta Therapeutics: Equity Ownership; Celgene: Consultancy; Cellworks Group, Inc.: Membership on an entity's Board of Directors or advisory committees; NeoImmune Tech: Research Funding; Amphivena Therapeutics: Consultancy, Research Funding; Bioline Rx: Research Funding, Speakers Bureau; Macrogenics: Research Funding, Speakers Bureau; Incyte: Consultancy, Research Funding; RiverVest Venture Partners Arch Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees. Liesveld:Onconova: Other: Data safety monitoring board; Abbvie: Membership on an entity's Board of Directors or advisory committees.

Background:The prevalence of obstructive sleep apnea syndrome (OSA) in children with sickle cell disease (SCD) is higher than in the general pediatric population. Adenotonsillectomy involves significantly increased risks in patients with SCD because of the need for general anesthesia,which could induce a vaso-occlusive event. Various preoperative regimens have been suggested to reduce peri-operative risks for SCD patients. Objective: We retrospectively reviewed the perioperative management for pediatric SCD patients undergoing adenotonsillectomy. We sought to identify the optimal preoperative hemoglobin (Hb) level for patients to have lowest risk of post-operative complications. Methods: Children between 1 and 18 years old with SCD including HbSS, HbSC, HbS beta thalassemia (Sβ0 and Sβ+) who underwent adenotonsillectomy from 2007 to 2017 were identified at Holtz Children's Hospital. Patients without these diseases who underwent the procedures were collected as a control. The study was approved by the University of Miami Institutional Review Board (IRB). De-identified data were exported to GraphPad Prism version 5.02 to perform the statistical analyses. Means were analyzed by student t test, rates by Chi-squared test. p < 0.05 is considered statistically significant. Results: Thirty-four patients with SCD (mean 7.1 yrs) and 145 controls (mean 7.0 yrs) were identified with adenotonsillectomy (see Table 1). SCD patients had significant longer hospital stay (2.6 versus 0.9 days, p< 0.001), and higher postoperative complication rates than the control group (20.6% vs 4.0%, p= 0.02). Most of the sickle cell patients' complications were hypoxemia (5 out of 7), except one supraventricular tachycardia and one headache. Four out of 7 patients with pretransfusion (pretrx) Hb level above 10 g/dL received manual exchange transfusion to keep Hb over 10 g/dL. None had complication or PICU stay. When patients' pretrx Hb levels was 9-10 g/dL, 5/8 received transfusion without significant complications, with the goal of not increasing post transfusion Hb over 11.5 g/dL. Most patients with pretrx Hb level 7-9 g/dL received simple blood transfusion. For patients with preoperative Hb level 9-11.5 g/dL, the PICU transfer rate was significantly lower than ones with Hb = 11.5 g/dL (11.5% vs 60%, p=0.04; see Figure 1). Complication rates were also lower in the Hb 9-11.5 g/dL group, but without statistical difference (19.2% vs 40%, p=0.3). Data is limited by small sample size in the SCD group. Conclusion(s): We concluded that for pediatric SCD patients planning for adenotonsillectomy, it is better to keep the posttransfusion Hb level at least above 9 mg/dL but less than 11.5 g/dL before procedure to avoid significant post operational complications. If pretransfusion Hb level is above 9 mg/dL, exchange transfusion by partial phlebotomy or simple transfusion (not to exceed 11.5 g/dL) may be considered. Disclosures No relevant conflicts of interest to declare.

Increasing evidence indicates that malignant stem cells are important for the pathogenesis of acute myelogenous leukemia (AML) and represent a reservoir of cells that drive the development of AML and relapse. Therefore, new treatment regimens are necessary to prevent relapse and improve therapeutic outcomes. Previous studies have shown that the sesquiterpene lactone, parthenolide (PTL), ablates bulk, progenitor, and stem AML cells while causing no appreciable toxicity to normal hematopoietic cells. Thus, PTL must evoke cellular responses capable of mediating AML selective cell death. Given recent advances in chemical genomics such as gene expression-based high-throughput screening (GE-HTS) and the Connectivity Map, we hypothesized that the gene expression signature resulting from treatment of primary AML with PTL could be used to search for similar signatures in publicly available gene expression profiles deposited into the Gene Expression Omnibus (GEO). We therefore devised a broad in silico screen of the GEO database using the PTL gene expression signature as a template and discovered 2 new agents, celastrol and 4-hydroxy-2-nonenal, that effectively eradicate AML at the bulk, progenitor, and stem cell level. These findings suggest the use of multicenter collections of high-throughput data to facilitate discovery of leukemia drugs and drug targets.

Fibrinogen BβArg448Lys is a common polymorphism, positioned within the carboxyl terminus of the Bβ-chain of the molecule. Studies suggest that it is associated with severity of coronary artery disease and development of stroke. The effects of the amino acid substitution on clot structure remains controversial, and the aim of this study was to investigate the effect(s) of this polymorphism on fibrin clot structure using recombinant techniques. Permeation, turbidity, and scanning electron microscopy showed that recombinant Lys448 fibrin had a significantly more compact structure, with thin fibers and small pores, compared with Arg448. Clot stiffness, measured by means of a novel method using magnetic tweezers, was significantly higher for the Lys448 compared with the Arg448 variant. Clots made from recombinant protein variants had similar lysis rates outside the plasma environment, but when added to fibrinogen-depleted plasma, the fibrinolysis rates for Lys448 were significantly slower compared with Arg448. This study demonstrates for the first time that clots made from recombinant BβLys448 fibrinogen are characterized by thin fibers and small pores, show increased stiffness, and appear more resistant to fibrinolysis. Fibrinogen BβArg448Lys is a primary example of common genetic variation with a significant phenotypic effect at the molecular level.

Acute myelogenous leukemia (AML) is a heterogeneous disease consisting of a variety of different leukemic subtypes. While acute promyelocytic leukemia displays marked sensitivity to the differentiating effects of trans-retinoic acid (tRA), other subtypes of AML display resistance. We now describe a novel compound (E)-4-[3-(1-adamantyl)-4-hydroxyphenyl]-3-chlorocinnamic acid (3-Cl-AHPC/MM002) that induces apoptosis in the tRA-resistant leukemia cell lines M07e, KG-1, and HL-60R, and in tRA-resistant patient leukemic blasts. The 3-Cl-AHPC totally inhibits leukemia colony formation at concentrations that inhibit committed human bone marrow stem cell proliferation, that is, granulocyte/macrophage colony-forming units (CFU-GMs) by only 30%. Exposure to 3-Cl-AHPC results in caspase activation and the cleavage of poly(adenosine diphosphate) (poly(ADP)) ribose polymerase. While activation of the extracellular signal-regulated kinase (ERK) and p38 pathways is not necessary for 3-Cl-AHPC-mediated apoptosis, maximal apoptosis requires c-Jun N-terminal kinase (JNK) activation. The 3-Cl-AHPC-mediated cleavage of the antiapoptotic B-cell leukemia XL (Bcl-XL) protein to a proapoptotic 18-kDa product is found in both the M07e cell line and patient leukemic blasts. The 3-Cl-AHPC treatment of mice bearing the AML 1498 cell line results in a 3.3-log kill in the leukemic blasts. While 3-Cl-AHPC does not activate retinoic nuclear receptors, it is a potent inducer of apoptosis in AML cells and may represent a novel therapy in the treatment of this disease.

Engraftment potential of hematopoietic stem cells (HSCs) is likely to be dependent on several factors including expression of certain adhesion molecules (AMs) and degree of mitotic quiescence. The authors investigated the functional properties and engraftment potential of Sca-1(+)lin(-) cells subfractionated on the basis of expression, or lack thereof, of CD11a, CD43, CD49d, CD49e, or CD62L and correlated that expression with cell cycle status and proliferative potential of engrafting fractions. Donor-derived chimerism in mice receiving CD49e(+) or CD43(+) Sca-1(+)lin(-) cells was greater than that in mice receiving cells lacking these 2 markers, while Sca-1(+)lin(-) cells positive for CD11a and CD62L and bright for CD49d expression mediated minimal engraftment. AM phenotypes enriched for engraftment potential contained the majority of high proliferative potential-colony forming cells, low proliferative potential-colony forming cells, and cells providing rapid in vitro expansion. Cell cycle analysis of AM subpopulations revealed that, regardless of their bone marrow repopulating potential, Sca-1(+)lin(-) AM(-) cells contained a higher percentage of cells in G(0)/G(1) than their AM(+) counterparts. Interestingly, engrafting phenotypes, regardless of the status of their AM expression, were quicker to exit G(0)/G(1) following in vitro cytokine stimulation than their opposing phenotypes. When engrafting phenotypes of Sca-1(+)lin(-) AM(+) or AM(-) cells were further fractionated by Hoechst 33342 into G(0)/G(1) or S/G(2)+M, cells providing long-term engraftment were predominantly contained within the quiescent fraction. These results define a theoretical phenotype of a Sca-1(+)lin(-) engrafting cell as one that is mitotically quiescent, CD43(+), CD49e(+), CD11a(-), CD49d(dim), and CD62L(-). Furthermore, these data suggest that kinetics of in vitro proliferation may be a good predictor of engraftment potential of candidate populations of HSCs. (Blood. 2000;96:1380-1387)