ALBERT

Description: Hemophilia is a genetic disease due to the deficiency of blood coagulation factor VIII or IX. Bleeding is common, especially into joints where a proliferative synovitis develops resulting in a debilitating arthritis, hemophilic arthropathy. The pathogenesis of blood-induced hemophilic synovitis (HS) is poorly understood. Repeated episodes of bleeding induce synoviocyte hypertrophy and hyperplasia, an intense neovascular response and inflammation of the synovial membrane. The component(s) in blood that initiates these changes is (are) not known, although iron is often proposed as one possibility. Previously, we reported that iron promotes the proliferation of human synovial fibroblasts and increases c-myc expression in vitro (Blood2002; 100(3)912–6). Here, we describe the proliferative effects of joint bleeding following traumatic hemarthrosis in a murine model of severe, human hemophilia A. Controlled, blunt trauma to the right knee joint consistently resulted in joint swelling due to a combination of bleeding and inflammation. The left, control knee was sham injured and remained normal for the duration of the experiment. Hemarthrosis resulted in acute morbidity evidenced by inactivity, weight loss and immobility. Hemosiderin was found in the synovial membrane of the right knees only. The numbers of synovial cell layers present in the membrane of the right (injured) knee increased during the experiment (figure). Expression of Ki67 in the nucleus, a marker of cell proliferation, confirmed the increase in synovial cell proliferation in membranes from the right (injured) knees. Taken together these data support the notion that iron causes aberrant gene expression and is, at least in part, responsible for the pathological findings observed in the joints of patients following bleeding and contributes to the development of hemophilic synovits. Figure Figure

Description: Discovery of somatic mutation of JAK-2 (G1849T that produces JAK-2V617F) in the hematopoietic cells of patients with Philadelphia chromosome negative myeloproliferative disorders (Ph−MPDs) was a watershed event that not only provided new insights into the pathobiology of polycythemia vera, essential thrombocytosis and primary myelofibrosis but also identified a potential target for therapy. Herein we report the results of preclinical studies designed to characterize the activity of a novel inhibitor of JAK-2. The compound, SGI-1252, developed by SuperGen (Dublin, CA) incorporates with high affinity into the ATP-binding site of JAK-2. SGI-1252 was tested against a panel of 75 kinases and was found to have significant activity against only FLT-3, TYK-2 and the SRC family members, ABL, LCK, YES, in addition to JAK-2 and JAK-1. SGI-1252 has an IC50 for JAK-2 of 5.4 nM with an IC50 for JAK-2V617F of 19.7 nM. The inhibitor also effectively blocks the activity of JAK-1 (IC50 14.8 nM) but has little JAK-3 inhibitory activity (IC50 1,700 nM). SGI-1252 is a potent inhibitor of STAT-5 phosphorylation (EC50 76.2 nM) and was also found to block the JAK-2 dependent expression of the anti-apoptotic protein, BCL-XL (EC50 778 nM). Drug treatment of a murine cell line (FDCP) transfected with either human wild-type JAK-2 or JAK-2G1849Tgenerated IC50 values of 83 nM and 108 nM, respectively, and SGI-1252 treatment of human cell lines, HEL, UKE-1 and SET-2, that express mutant JAK2 in different copy numbers, gave IC50 values of 472 nM, 83 nM and 63 nM, repectively. When tested in ex-vivo expanded native human erythroid progenitor cells from 17 patients with Ph−MPDs (10 PV and 7 MF), SGI-1252 showed an IC50 of ~100 nM, regardless of the JAK-2V617F allele burden. Using a flow cytometric assay, SGI-1252 was shown to induce apoptotic cell death in a concentration dependent manner. Luminex technology allows for concurrent quantitative analysis of multiple proteins from the same tissue source, and this technology was used to investigate simultaneously the effects of SGI-1252 on total and phospho ERK1/2, total and phospho STAT3, phospho STAT5, caspase 3, cleaved PARP and GAPDH (control) in untreated and drug treated cells at IC50 and IC80 concentrations. Significant in vivo efficacy of SGI-1252 was also observed using HEL and MV-4-11 xenograft models when compared to treatment with vehicle or daunorubicin. Using a murine model, we found that SGI-1252 has high oral bioavailability and is well tolerated with a five-day repeat maximum dose of at least 900 mg/kg. Together, these studies demonstrate that SGI-1252 is a potent inhibitor of JAK-2 dependent proliferation in both JAK-2V617F positive cell lines and in ex vivo expanded erythroid progenitors derived from patients with JAK-2V617F positive Ph−MPDs. Moreover, our studies show that the effects of SGI-1252 are mediated by blocking both JAK-2 dependent anti-apoptoic pathways and JAK-2 dependent proliferative pathways. Using the orally available form of the compound, pharmacokinetic, pharmacodynamic and toxicity studies in mice suggest that serum concentration of the drug well above the predicted therapeutic range can be achieved without significant hematological toxicity. Based on these preclinical experiments, SGI-1252 appears to be an excellent candidate for phase I/II studies in patients with Ph−MPDs.

Description: Introduction: Alterations of innate immunity signaling and activation of NF-κB are known to occur in low/int-1-risk MDS (Braun, 2006, Wei, 2013). These suggest that modulation of NF-κB could be a therapeutic target in this group of patients. Bortezomib is a proteosome inhibitor with potential inhibitory activity against NF-κB. To determine whether bortezomib has therapeutic activity in lower-risk MDS patients, we designed a phase II trial of SC bortezomib in patients with lower-risk MDS and evidence of NF-κB activation. Methods: This was a single-arm phase II study of bortezomib in low/int-1-risk MDS patients. Bortezomib was administered at 1.3 mg/m2 SC on days 1, 4, 8, and 11 in a 21 day cycle for a maximum of 2 years of therapy. Eligibility criteria included age ≥18 years, adequate performance and organ function, and having received at least 1 prior therapy. Patients with grade 2 or greater peripheral neuropathy at baseline were excluded. Patients were then prescreened prospectively for enrollment by assessing cellular levels of the phosphorylated NF-κB subunit p65 (pp65) in their marrows. This was performed in bone marrow aspirate smears stained for immunofluorescence using an antibody against a phosphorylated form of NF-κB p65 (phospho-Ser276). This assay was performed in the Department of Hematopathology following CLIA regulations. Pp65 was considered positive if at least 5% of all the nucleated marrow cells were positive for pp65 staining. Subsequently, pp65 levels were assessed on day 21 of cycles 1 and 2 and then in subsequent marrows as clinically indicated. Responses were assessed according to IWG06. The study could accrue a maximum of 40 patients if there were at least a 95% chance of at least a 15% ORR. Results: Since 9/2013, we have enrolled 12 patients with a median age of 72 years (range 56 - 87). Median marrow blast percentage was 1.5% (range 1 – 5%). Nine patients had diploid cytogenetics, 2 had del 20q, and 1 had trisomy 8. All patients had low (2 patients) or int-1 (10 patients) disease. All were transfusion dependent (6 both platelets and red cells (PRBCs), 1 only platelets and 5 only PRBCs). Ten (83%) of the 12 had received prior hypomethylating agent (HMA) therapy. Thus far, patients have received a median of 3.5 cycles (range 1-12). Baseline median pp65 was 26.5% (range 7 – 70%). Five patients were taken off the study, 2 for disease progression and 3 for no response after a median of 3 cycles. Seven remain on study. No grade 3/4 toxicity and minimal grade 1/2 toxicity have been observed. No peripheral neuropathy has been observed. The median overall survival has not been reached. At a median follow up of 17 weeks, the overall response rate is 33% (3 HI-EP and 1 HI-N). Six patients have stable disease, and 2 had progression. One patient achieved red cell transfusion independence, going from PRBCs every 2 weeks to no transfusions after 2 cycles. All responders had been previously treated with an HMA. At day 21, the median pp65 level was 23% (0-70) and subsequently 22% (5-50) (all NS). Pp65 decreased by at least 50% in 5 patients, but it fell below 5% in only 2 patients. Two of the 5 patients with decreased levels of pp65 had a response to therapy. Of interest, the patient that achieved transfusion independence had a substantial decrease in pp65 (to less than 5%) that was lost (up to 56% pp65) when the patient lost hematological response. We also screened patients with a 28-gene mutation panel and found a MET, JAK2, or TET2 mutation in each of the 3 patients achieving a hematological improvement, a RUNX1 or CEBPA in each of the two patients with progressive disease, and an APC or ASXL1 mutation in each of the patients with stable disease. Conclusions: Bortezomib is well tolerated and results in hematological improvement in pretreated patients with lower-risk MDS. It is feasible to assess pp65 in serial bone marrow samples in this group of patients. Pp65 decreases in 40% of patients, and substantial decreases may be associated with response. This study suggests that it is possible to target patients for therapeutic intent using the NF-κB pathway as a biomarker. Disclosures No relevant conflicts of interest to declare.

Description: Hemophilia is a genetic disease caused by a deficiency of blood coagulation factor VIII or IX. Bleeding into joints is the most frequent manifestation of hemophilia. Hemarthrosis results in an inflammatory and proliferative disorder termed hemophilic synovitis (HS). In time, a debilitating, crippling arthritis, hemophilic arthropathy, develops. Although the clinical sequence of events from joint bleeding to synovitis to arthropathy is well documented, the component or components in blood and the molecular changes responsible for hemophilic synovitis are not known. Iron has long been suspected to be the culprit but direct evidence has been lacking. Previously, we showed that iron increased human synovial cell proliferation and induced c-myc expression. Here we show that bleeding into a joint in vivo and iron in vitro result in increased expression of the p53-binding protein, mdm2. Iron induced the expression of mdm2 by normal human synovial cells approximately 8-fold. In a murine model of human hemophilia A, hemarthrosis resulted in pathologic changes observed in human hemophilic synovitis and a marked increase in synovial cell proliferation. Iron, in vitro, induced the expression of mdm2. The molecular changes induced by iron in the blood may be the basis of the increase in cell proliferation and the development of hemophilic synovitis. (Blood. 2004;104:2060-2064)

Description: Key Points CD37 positivity predicts significantly better survival for DLBCL, and is superior to other prognostic factors in GCB-DLBCL. CD37 loss is an important risk factor for R-CHOP resistance in both GCB- and ABC-DLBCL.

Description: Introduction MDS has been linked to constitutive activation of genes involved in the nuclear factor-kappaB (NF-κB) pathway [Wei et al. Leukemia 2013; Braun et al. Blood 2006]. Hence, NF-kB is an attractive therapeutic target in this disease. Bortezomib is a proteosome inhibitor with inhibitory activity against NF-κB. We designed a phase II trial of SC bortezomib in patients with low- to intermediate (low/int-1)-risk MDS and evidence of NF-κB activation to determine its therapeutic activity in these patients. Methods In this single-arm phase II study in low/int-1-risk MDS patients, bortezomib was administered at 1.3 mg/m2 SC on days 1, 4, 8, and 11 in a 21-day cycle for a maximum of 2 years of therapy. Eligibility criteria included age ≥18 years, adequate performance status and organ function, and having received at least 1 prior therapy. Patients with grade 2 or greater peripheral neuropathy at baseline were excluded. Patients were prescreened prospectively for enrollment by assessing cellular levels of the phosphorylated NF-κB subunit p65 (pp65) in their marrow. This was performed by immunofluorescence with phospho-Ser276 in bone marrow aspirate smears in the Department of Hematopathology (CLIA regulations). Patients were eligible if at least 5% of all nucleated marrow cells were positive for pp65 staining. pp65 levels were assessed again on day 21 of cycles 1 and 2 and then as clinically indicated. Responses were assessed according to IWG [Cheson et al. Blood 2000; Cheson et al, Blood 2006]. The study could accrue a maximum of 40 patients if there were at least a 95% chance of at least a 15% ORR. Results Beginning 9/2013, we enrolled 15 patients with a median age of 71 years (range 56 - 87). Median marrow blast percentage was 1.9% (range 0 - 5%). Eleven patients (73%) had diploid cytogenetics, 2 had del(20q), 1 had Y-, and 1 had del(5q). All had lower-risk MDS by IPSS (low 33.3%, int-1 66.7%). All were transfusion dependent (6 both platelets and red cells (PRBCs), 1 only platelets, 8 only PRBCs). Hypomethylating agents had failed in 12 of the patients. At a median follow-up interval of 22 weeks, the ORR was 20%; 3 patients had hematologic improvement with a mean duration of response of 14.3 weeks (range 4-21). Eight patients had stable disease, and 4 had progression. No correlation between clinical response and molecular or cytogenetic data was observed. Eventually, all patients were taken off study: 7 due to increasing transfusion requirements, 2 worsening cytopenia, 1 lack of response, 1 increased blasts, and 1 grade 2 neuropathy; 2 withdrew by choice, and 1 patient died from causes unrelated to the study. Four patients experienced ≥ 1 grade 3 toxicity. No grade 4 toxicity was observed. Seven patients experienced grade 1 (n=4) or grade 2 (n=3) neuropathy. Morphologic review (n=14) in the responders group (n=3) showed reduction in ring sideroblasts (RS) in 2 patients (60 to 47% and 43% to 30%). Two patients, including 1 of those with reduced RS, had improvement of dysplasia (from severe trilineage dysplasia to moderate bilineage dysplasia). No changes in cytogenetic studies were found in the responder group. Among nonresponders (n=11), 3 had new acquired cytogenetic abnormalities, 3 had worsening dysplasia, and 2 had increase in blast count (1% to 3% and 1% to 4%). Two had no morphologic changes during treatment. One showed improvement in dysplasia and blast count (2% to 0%) and no changes in cytogenetics. Interestingly, 3 nonresponders showed RS reduction during treatment (18% to 1%, 85% to 45%, and 6% to 0%). The median pp65 level at baseline was 30.87% (range 7 - 70%). The pp65 level decreased in 7 of the 15 patients (46.7%), in 6 of them by the end of cycle 1. Interestingly, the 3 responders were among the 7 patients who had a decrease in pp65 level. Eventual loss of response in these patients was accompanied by return to a higher pp65 level. In nonresponders, the pp65 level increased in 7, decreased in 2, and remained unchanged in 1; for 1, the sample was suboptimal. Conclusions In previously treated lower-risk MDS patients, SC Bortezomib was well tolerated and resulted in hematologic improvement and decrease in RS. NF-κB activation, measured by pp65 level, can be a useful biomarker to select patients with lower-risk MDS who could benefit from therapies that target this pathway. The NF-κB pattern of expression suggests an inverse relationship between treatment response and NF-κB level, with associated improvement in bone marrow morphology. Disclosures Konopleva: Calithera: Research Funding; Cellectis: Research Funding. Cortes:ARIAD: Consultancy, Research Funding; Bristol-Myers Squib: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Research Funding.

Description: Gemtuzumab ozogamicin (GO) is an FDA-approved antibody-drug conjugate that has shown clinical benefit in acute myeloid leukemia (AML) patients but has been voluntarily withdrawn from the market in the US. GO targets CD33 on AML tumor cells and delivers its cytotoxic payload, namely calicheamicin. Despite promising initial responses to GO, many patients relapsed while on therapy, and the development of clinical resistance to GO hampers its effectiveness. Initial investigations into the mechanisms responsible for clinical resistance to GO implicated the activity of multidrug resistance proteins (MDR) as a major contributor to the lack of clinical efficacy. To better understand the molecular mechanisms that may drive resistance to the payload used in GO, as well as standard of care agents in AML, we created calicheamicin-resistant and cytarabine-resistant AML cell lines. HL60 cells were continuously exposed to increasing concentrations of calicheamicin or cytarabine until resistant populations emerged. Calicheamicin-resistant HL60 (HL60-CAL-R) cells are ~40-fold resistant to calicheamicin and cytarabine-resistant HL60 (HL60-CYTAR-R) are ~2000- fold resistant to cytarabine compared to their isogenic parental counterparts as determined by in vitro cytotoxicity assays. Initial model characterization of HL60-CAL-R suggests that continuous exposure to calicheamicin induces the acquisition of a chemo-resistant phenotype highlighted by increased expression of MDR1 and MRP1/2. Furthermore, functional assays by flow cytometry revealed high levels of efflux activity in MDR1 and MRP1/2, but not BCRP, in HL60-CAL-R as compared to parental HL60. HL60-CAL-R could be re-sensitized to calicheamicin with the addition of verapamil (MDR1 inhibitor) or reversan (MDR1 and MRP1/2 inhibitor). HL60-CAL-R shows cross-resistance to daunorubicin (MDR1 substrate), but not cytarabine (non-MDR1 substrate), supporting MDR1 upregulation as one major mechanism of resistance to calicheamicin in this cell line. HL60-CAL-R cells are also cross-resistant to GO. In contrast, HL60-CYTAR-R do not have an induction of MDR1 and MRP1/2 protein levels or activity and retain sensitivity to GO in vitro and in vivo. Initial RNA-Seq profiling of HL60-CYTAR-R cells suggest they have decreased DCK expression, the loss of which was previously shown to mediate cytarabine resistance in other models. Intriguingly, HL60-CAL-R and HL60-CYTAR-R cells retained sensitivity to a proprietary next-generation DNA alkylating- and crosslinking- agent being developed at Pfizer, cyclopropylpyrrolo[e]indolone (CPI). These data demonstrate the utility of generating and characterizing drug-resistant cell lines to uncover clinically relevant mechanisms of resistance and identify next-generation compounds that can overcome them. Disclosures Kahler: Pfizer: Employment. Dougher:Pfizer: Employment. Lu:Pfizer: Other: Ex-Pfizer employee. Xu:Pfizer: Employment. Kausar:Pfizer: Other: Ex-Pfizer Employee. Lemon:Pfizer: Employment. Zhong:Pfizer: Employment. Lucas:Pfizer: Employment. Sung:Pfizer: Employment. Sapra:Pfizer: Employment, Equity Ownership.