bluuebunny/crossref_metadata_2025 huggingface dataset:
Incidence and Outcomes of Myeloproliferative Neoplasms (MPN) in Adolescents and Young Adults (AYAs).
Authors: Pemmaraju Naveen , Kantarjian Hagop M , Cortes Jorge E. , Quintas-Cardama Alfonso , Pierce Sherry A. , Verstovsek Srdan
Published: October 2019

Abstract 2845

Background:

The MPNs are a family of chronic hematologic malignancies that typically affect pts ages 60–70s. However, there is growing awareness of hematologic malignancies developing in young pts, particularly a unique subset of pts known as AYAs. Little is known about incidence and outcomes of AYA pts with MPNs.

Objectives:

To determine the incidence and outcomes of MPN AYA pts.

Results:

We retrospectively reviewed charts of 1,616 MPN pts evaluated at our institution from 1986–2011. A total of 171 MPN pts (11%) were identified whose ages were 16–39, defined as AYA pts (per NCCN guideline recommendations on AYA cancers). Breakdown by MPN subtype: Essential Thrombocytosis, ET (n=78, 46% of AYA MPNs), Polycythemia Vera, PV (n=25, 15%), Myelofibrosis, MF(n=24, 14%), [3 post-ET, 1 post-PV, 20 primary MF], Hypereosinophilic syndrome, HES (n=22, 13%), and Systemic Mastocytosis, SM (n=22, 13%). Baseline characteristics of AYA MPN pts detailed in Table 1. Only significant difference among AYA and non-AYA pts in terms of baseline CBC parameters was WBC: 6.4 (0.4–108.4) in AYA MF vs 10.1 (0.6–361) in non-AYA MF, p=0.0049. Analysis of median overall survival (OS), by Kaplan-Meier method, compared by age group (AYA vs non-AYA pts) and broken down by MPN subtype, shown in Table 2.

Conclusion:

MPN AYA pts constitute 11% of MPN pts at our institution. Overall, their 5- and 10- yr OS were significantly better than their older counterparts in the 3 major MPN subtypes (ET, PV, MF) and trended towards better survival but not statistically significant in HES and SM. Among AYA MPN pts, female predominance was noted among ET and SM pts and only 1 transformation event was noted among all MPN AYA pts in this analysis.

Disclosures:

No relevant conflicts of interest to declare.
Signal Transducer and Activator of Transcription (STAT)-3 Induces the Expression of Microrna-155 in Chronic Lymphocytic Leukemia (CLL) Cells.
Authors: Li Ping , Grgurevic Srdana , Liu Zhiming , Harris David , Rozovski Uri , Keating Michael J. , Calin George , Estrov Zeev
Published: October 2019

Abstract 2885

MicroRNAs (miRs) are involved in the initiation, progression and dissemination of CLL cells (Calin GA, Croce CM. Blood 114:4761, 2009). Recent studies showed that high levels of miR-155, previously shown to regulate hematopoietic cell development, are expressed in CLL cells. Because transgenic miR-155 overexpression in the mouse stimulates B-cell proliferation, it is thought that miR-155 plays a role in the pathogenesis of CLL (Calin GA et al. N Engl J Med 353:1793, 2005). STAT3 is constitutively activated in CLL and induces the transcription of several STAT3-regulated genes. A recent study demonstrated that STAT3 activates miR-21 and miR-181b-1 (Iliopolus D. et al. Mol Cell 39:493, 2010). Therefor we wondered whether STAT3 enhances the expression of miR-155 in CLL cells. Because a sequence analysis revealed that the promoter of miRNA-155 harbors γ-interferon activation sequence-like elements typically activated by STAT3, we sought to determine whether STAT3 directly activates miR-155 expression. We generated truncated constructs of the miR-155 promoter, co-transfected them into MM1 cells together with STAT3 small interfering (si) RNA (siRNA), and assessed their luciferase activity. The luciferase activity data suggested that of the two putative STAT3 binding sites only one site is involved in STAT3 induced transcription because STATR3-siRNA reduced the activity of miRNA-155 promoter of constructs that harbor this site. To confirm these data we performed an electrophoretic mobility shift assay (EMSA) and chromatin immune-precipitation (ChIP). The EMSA confirmed that STAT3 bound the miR-155 promoter in fresh CLL cells, and ChIP confirmed that STAT3 bound one putative STAT3-binding site in the miR-155 promoter but not to the other, as demonstrated by the luciferase assay; STAT3 co-immuno-precipitated only one putative STAT3 binding region of miR-155 promoter and other STAT3-regulated genes. Finally, STAT3-small hairpin RNA (shRNA) downregulated miR-155 and other STAT3-regulated genes, suggesting that constitutively activated STAT3, binds miR-155 promoter and induces miR-155 transcription in CLL cells.

Disclosures:

Keating: Celgene Corporation: Consultancy, Research Funding; Roche: Consultancy, Research Funding; Xcenda: Consultancy, Speakers Bureau.
Discovery and Characterization of Chemical Inhibitors of UBC13.
Authors: Madiraju Charitha , Matsuzawa Shu-ichi , Ardecky Robert , Pass Ian , Ngo Tram , Matsuzawa Yasuko , Wimer Carina , Cuddy Michael , Diaz Paul , Ricardo Correa , Tamble Craig , Shu Chih-Wen , Pinkerton Anthony , Sergienko Eduard , Hassig Chris , Chung Thomas , Reed John C.
Published: October 2019

Abstract 2950

Poly-ubiquitination of signaling proteins via lysine 63 (K63)-linked chains is recognized as a critical post-translational modification involved in activation of NF-kB and stress kinases in the context of signaling by Tumor Necrosis Factor Receptors (TNFRs), Toll-like receptors (TLRs), and antigen receptors. UBC13 is a K63-specific ubiquitin conjugating enzyme that partners with TNFR-associated factors (TRAFs) to mediate K63-linked ubiquitination. Gene ablation studies have shown UBC13 is required for NF-kB signaling induced by a variety of stimuli in specific types of immune cells, making it a potential target for certain cancers, autoimmune and inflammatory diseases. UBC13 operates together with obligatory cofactors, either UEV1A in the cytosol or MMS2 in the nucleus. The nuclear function of UBC13 is evolutionarily conserved, where it plays a critical role in double strand DNA repair, making UBC13 a potential chemo- and radio-sensitizer target for oncology.

To identify chemical inhibitors of UBC13, we developed a HTS assay measuring UBC13-UEV1A enzymatic activity by TR-FRET, screening altogether ∼450,000 diverse compounds. Hit compounds were characterized using a rigorous testing funnel consisting of (a) informatics filtering against a database of > 100 HTS campaigns conducted with the same libraries, to eliminate promiscuous compounds; (b) counter-screens against E1, another cysteine-dependent enzyme (caspase-3), and against an irrelevant target formatted as a TR-FRET assay; and (c) ordering compounds from fresh powders and demonstrating reproducible concentration-dependent inhibition of UBC13. The surviving hits were then analyzed by cell-based assays for suppression of TRAF6 ubiquitination but not Mdm2-mediated ubiquitination of p53, resulting in 14 promising hits that included two chemical series. While suppressing TRAF6 ubiquitination (UBC13-dependent) in cells, these compounds did not interfere with either SUMOylation (UBC9-dependent) or NEDDylation (UBC12-dependent) of cellular proteins. UBC13 inhibitors also suppressed NF-kB activity (measured using stably integrated NF-kB-driven luciferase reporter gene) induced by PKC activators (Carma/Bcl-10/MALT pathway) and DNA damaging agent (Doxorubicin) but not by TNF-a. Investigations of the bioactivity of these UBC13 inhibitory compounds and their analogs will be described for a variety of hematolymphoid malignancies. (Supported by NIH R03-MH085677, NIH U54–005033, and by a fellowship grant from International Myeloma Foundation).

Disclosures:

No relevant conflicts of interest to declare.
Inhibition of Wnt/ß-Catenin Pathway with ICG-001 Induces Apoptosis in Multiple Myeloma.
Authors: Grigson Eileen R , Pisklakova Alexandra , Weissman Brittany , Nefedova Yulia
Published: October 2019

Abstract 2951

Multiple Myeloma (MM) is characterized by clonal expansion of malignant plasma cells, which preferentially reside in the bone marrow (BM). It has been previously shown that canonical Wnt signaling plays a critical role in proliferation and differentiation in MM. ICG-001 is a selective Wnt/ß-catenin signaling inhibitor that binds to the amino terminus of CREB, thus preventing ß-catenin/TCF signaling in the nucleus. Here we investigated whether the pharmacological inhibition of canonical Wnt signaling with ICG-001 could affect viability of MM cells and produce anti-myeloma effect.

C-myc and cyclin D1 are known to be downstream target genes of canonical Wnt signaling. MM cells lines treated with ICG-001 revealed down regulation of c-myc and cyclin D1 as compared to control. To investigate the varying sensitivity of cell lines to ICG-001, four cells lines were used: RPMI-8226, NCI-H929, U266, and MM1S. MM cells lines were treated with ICG-001 (1uM – 40uM) and data was collected using MTT assay or flow cytometry (Annexin binding assay). ICG-001 significantly reduced viability in all four cell lines due to induction of apoptosis, with RPMI-8226 showing the greatest sensitivity. We performed cleaved-caspase assays and demonstrated that apoptosis was caspase-3 dependent. We have also evaluated the effect of ICG-001 in BM samples from patients with MM. BM cells were treated with ICG-001 for 24 hours and the level of cleaved-caspase-3 was evaluated by flow cytometry. MM cells were defined as double positive CD138+ and Lambda/Kappa+. Our results demonstrated that MM cells were sensitive to ICG-001, while this drug was not able to induce cell death in non-tumor BM cells.

To test the effect of ICG-001 in vivo we injected SCID/Beige mice with 10 × 105 8226 cells in 100 μL phosphate-buffered saline (PBS). After 3 weeks when a tumor was measurable, the mice were split into two groups and were treated with either ICG-001 (100 mg/kg) or vehicle control (PBS) daily, for 14 days. Tumor size was consistently monitored during treatment and 3 weeks after treatment. We observed that ICG-001 produced a significant antitumor effect as compared to control group.

These data indicate targeting Wnt/ß-catenin Pathway with ICG-001 could be therapeutically beneficial to patients with MM.

Disclosures:

No relevant conflicts of interest to declare.
Identification of Multiple Subclones in Peripheral T-Cell Lymphoma, Not Otherwise Specified with Genomic Aberrations
Authors: Yoshida Noriaki , Umino Akira , Liu Fang , Arita Kotaro , Karube Kennosuke , Tsuzuki Shinobu , Ohshima Koichi , Seto Masao
Published: October 2019

Abstract 298

Peripheral T-cell lymphoma, not otherwise specified (PTCL, NOS) is a heterogeneous group of nodal and extra-nodal mature T-cell lymphomas that do not belong to any of the hitherto characterized T cell lymphoma subtypes. Adult T-cell leukemia/lymphoma (ATLL) is a mature T-cell neoplasm caused by human T-cell leukemia virus type 1 (HTLV-1). PTCL, NOS with genomic aberrations has been shown to resemble lymphoma-type ATLL in terms of genomic aberration patterns, histopathology and prognosis (Clin Cancer Res., 15:30–38). We have recently shown that a majority of patients with acute-type ATLL have multiple subclones that were likely produced in lymph nodes (Blood, 117:5473–5478). In this study, high-resolution oligo-array comparative genomic hybridization (CGH) was employed to determine whether PTCL, NOS with genomic aberrations also possesses multiple subclones as found in ATLL. Thirteen cases of PTCL, NOS were available for 44K high-resolution array CGH analysis. These samples analyzed in this study were shown to be monoclonal by Southern blot analysis. We obtained average log2 ratios of regions where aberrations continued over 400 probes and showed a linear average ratio of >0.1 or <-0.1. The log2 ratio imbalance was defined when a patient had a different average log2 ratio for gain or loss regions. The results showed that 11 of the 13 cases (84.6%) had a log2 ratio imbalance, suggesting that multiple subclones exist in PTCL, NOS with genomic aberrations. As previously demonstrated, the average log2 ratio of aberrant regions reflected the ratio of tumors (Blood, 117:5473–5478). In one case, as represented in Figure 1, all tumor cells had regions of loss on chromosomes 3 and 13. Fifty-four percent of tumor cells had a region of loss on chromosome 16, and 83% of tumor cells had loss on chromosome 17. It is therefore speculated that clonal evolution took place as shown in Figure 2. In an effort to examine the association between multiple subclones and prognosis, we used our previous BAC array data for 24 cases and found that the presence of multiple subclones was associated with a poor prognosis (p=0.0279) (Figure 3). Our finding concerning the presence of multiple subclones in PTCL, NOS and ATLL should assist greatly in revealing the mechanism of lymphoma development, progression and drug-resistance associated with these diseases.

Disclosures:

No relevant conflicts of interest to declare.
Tissue-Specific Homing and Different Impact in Gvhd Prevention of NK Cell Subpopulations.
Authors: Meinhardt Kathrin , Bauer Ruth , Kroeger Irena , Schneider Julia , Ganss Franziska , Dudziak Diana , Rehli Michael , Mackensen Andreas , Ullrich Evelyn
Published: October 2019

Abstract 3004

Clinical studies exploiting the impact of natural killer (NK) cells in allogeneic hematopoietic stem cell transplantation (HSCT) have provided promising results. It is known that NK cells are a heterogeneous population and can be divided into functionally distinct NK cell subpopulations. Murine NK cells can be separated along their expression of CD27 and CD11b and CD117 (c-kit). However, the functional relevance of distinct NK cell subsets in graft-versus-host-disease (GVHD) has not been investigated in detail so far. We have established different protocols for ex vivo isolation and expansion of murine NK cell subpopulations. These NK subsets were further analyzed in vitro and in vivo in an allogeneic murine GVHD model. Here we report on different genomic, phenotypic and functional properties of 4 NK cell subsets. Our data clearly demonstrate that CD27+ NK cells revealed the highest IFN-g production upon coculture with tumor cells and/or IL-2.

Interestingly, the CD11b+ NK cells express multiple genes of cytotoxic pathways and develop the highest cytotoxic capacity towards tumor cells. We observed up to 60% tumor lysis by CD27- CD11b+ NK cells compared to 40–45% by CD27+ CD11b+, about 25% by CD27+ CD11b- and 10% by c-kit+ CD11b- NK cells at an effector-target ratio of 5:1, respectively. Furthermore, the CD11b+ NK cell subset significantly reduced T cell proliferation induced by allogeneic dendritic cells in mixed lymphocytes reactions.

Next, we analyzed the migratory capacity and tissue-specific homing of FACS-sorted NK cell subsets by adoptive transfer of congeneic CD45.1+ and Luc+ NK cell subpopulations in autologous and allogeneic bone marrow transplantation. Of interest, FACS analysis and in vivo imaging showed that CD11b+ NK cells migrated to peripheral GVHD target organs, whereas CD27+ NK cells preferentially homed to the bone marrow.

Finally, this study addressed for the first time the role of distinct NK cell subpopulations in the development of GVHD in a fully MHC mismatched HSCT mouse model. Importantly, we identified the CD11b+ NK cell population as the NK cell subset that significantly diminished GVHD. In vivo imaging of Luc+CD11b+ NK cells revealed that this subset migrates to the colonic tissue to prevent development of GVHD colitis as shown by colonoscopy.

In summary, our comparative study outlines that only CD11b+ NK cells, migrating to the peripheral GVHD target organs and providing the most efficient cytolytic capacity directed against allogeneic dendritic cells, protect against GVHD. These new insights are highly relevant for the selection of optimal NK cell subsets in the field of cellular immunotherapy.

Disclosures:

No relevant conflicts of interest to declare.
Allogeneic Cytokine-Induced Killer Cell-Dependent Eimination of Host Dendritic Cells Leads to Less Graft-Versus-Host Disease.
Authors: Mase Shintaro , Maeba Hideaki , Fujiki Toshihiro , Kuroda Rie , Araki Raita , Koizumi Shoichi , Yachie Akihiro , Nishimura Ryosei
Published: October 2019

Abstract 3011

Cytokine-induced killer (CIK) cells are ex vivo-expanded T lymphocytes expressing both natural killer (NK)- and T-cell markers. We have reported that adoptive transfer of allogeneic CIK cells in a murine model caused minimal graft-versus-host disease (GVHD) with retention of antitumor activity mediated by NKG2D, which is an activating receptor expressed on NK cells. The mechanism of suppression of GVHD with CIK cells is not fully understood. Host residual dendritic cells (DCs) are most important cells in initiating GVHD reaction. Therefore, we hypothesized that alloreactive NK cells, even when infused in large numbers, do not cause GVHD by killing recipient DCs with a same mechanism as CIK cells kill tumor cells. To test this, DCs generated from rodent bone marrow cells were used for 51-Cr release cytotoxicity assays as target cells. We demonstrated that though autologous CIK cells (Balb/c) had relatively strong killing activity against mature DCs (Balb/c), allogeneic CIK cells (B6) had much more killing activity even from at a 10:1 effector -target ratio as shown in Fig 1 below. In addition, killing activity against DCs did not changed with/without adding NKG2D blocking antibody, suggesting that there were possible mechanisms for cell killing other than NKG2D/NKG2D ligand system in CIK cells. To further evaluate whether allogeneic CIK cells eliminate host DCs to reduce GVHD in vivo, lethally irradiated Balb/c recipients were given BM (B6) with CIK cells or splenocytes to compare the number and activation status of residual host-typed DCs in the spleen after bone marrow transplantation. As shown in Fig 2, on day 1 after BMT absolute number of host DCs in spleen receiving splenocytes and CIK cells were almost same, however, the numbers of DCs in the mice receiving BM alone were much less. On day 3, absolute number of host DCs in all groups decreased. In particular, the number of host DCs in the mice receiving CIK cells dramatically decreased and finally reached to the same number of host DCs in the mice receiving BM alone. On the contrary, the number of host DCs in the mice receiving splenocytes didn't decrease so much and was significantly greater than number of host DCs in the mice receiving BM alone. Considering the above, when allo-CIK cells first met to DCs, both DCs and CIK cells were expanded on day 1 after BMT, and then allo-CIK cells turned to kill host DCs. In conclusion, allogeneic CIK cells caused less GVHD due in part to elimination of host DCs.

Disclosures:

No relevant conflicts of interest to declare.
Peripheral Blood CD34+ Specific Donor Chimerism Analysis As an Early Indicator of Disease Relapse After Allogeneic Hematopoietic Stem Cell Transplant in Patients with Myelodysplastic Syndromes and Acute Myelogenous Leukemia.
Authors: Norkin Maxim , Meacham Amy , Ta Mai H , Benson Neil , Goldstein Steven , Rosenau Emma , Hsu Jack W. , Cogle Christopher R. , Hiemenz John W , Brown Randy A. , Wingard John R.
Published: October 2019

Abstract 3074

Background:

Disease relapse after allogeneic hematopoietic stem cell transplant (HCT) remains a major obstacle to treatment success and cure for patients with myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML). Early identification of patients at a high risk for disease relapse has significant practical implications allowing early therapeutic interventions. We evaluated a novel approach for an early identification of a post-transplant relapse in patients with MDS and AML.

Methods:

CD34+ specific donor chimerism analysis was obtained in 6 HCT recipients with CD34+ AML and MDS. Starting at day +30 post HCT CD34+ cells from peripheral blood were positively selected by immunomagnetic isolation and then underwent fluorescence-activated cell sorting. Purified CD34+ cells were subsequently evaluated for percentage of donor DNA contribution by short tandem repeat (STR) analysis. CD34+ specific donor chimerism analysis was repeated monthly until hematologic relapse or death occurred. Simultaneously, conventional donor chimerism analysis was measured in the subpopulations of peripheral blood cells.

Results:

CD34+ cells were isolated from peripheral blood with a very high purity >95% and in sufficient quantity for reliable STR analysis in all study patients. Out of 6 evaluated patients, 3 developed hematologic relapse confirmed by bone marrow evaluation. In each relapsed patient CD34+ specific chimerism dropped to <15% prior to the confirmation of hematologic relapse. However, conventional STR analysis in granulocytes, T-cells and B-cells remained >80% (Figure 1). Moreover, obvious and steady decline in CD34+ specific donor chimerism significantly preceded the time of hematologic relapse. In contrast, no decline in CD34+ chimerism was identified in 3 patients without hematologic relapse. (Figure 1).

Conclusion:

Though this is a preliminary study, the results suggest that CD34+ specific donor chimerism analysis is a useful technique for timely identification of hematologic relapse in MDS and AML patients after HCT and may provide significantly earlier relapse detection than conventional chimerism analysis.

Disclosures:

No relevant conflicts of interest to declare.
Bortezomib-Based Induction Overcomes Effect of 1q Amplification On Response in Newly-Diagnosed Myeloma and Results in Similar 2 Year Event-Free Survival
Authors: Joshua Douglas E , Hertzberg Mark S , Auguston Bradley , Spencer Andrew , Horvath Noemi , Bashford John , Campbell Lynda J , Ashurst Eleanor , Wade Jacquelyn , Copeman Michael C. , Butcher Belinda E , Prince Miles
Published: October 2019

Abstract 4065

Background:

Amplification of chromosome 1q occurs in 40% of myeloma and predicts lower response to induction regimens. We assessed if a PAD regimen overcomes effects of 1q amplification on overall response rate (ORR), event-free (EFS) and overall survival (OS) in newly-diagnosed, transplant-eligible myeloma.

Methods:

This phase II trial enrolled 107 patients, stratified by 1q amplification. All could receive 4× 21-day cycles of PAD induction: bortezomib 1.3mg/m2 D 1,4,8,11; doxorubicin 20mg/m2 D1,4; dexamethasone 20mg D1,2,4,5,8,9,11,12. Responders proceeded to melphalan 200mg/m2 and ASCT. ORR after PAD was primary endpoint. Secondary endpoints included ORR 3 monthspost-ASCT, and 2-year EFS and OS.

Results:

22% (20/93 evaluable cases) had amplified 1q. Median PAD cycles was4. ORRs were similar in amplified and non-amplified cases (100% vs. 87% post-PAD; 100% vs. 93% post-ASCT). After median 2 years, 2-year EFS was similar in amplified and non-amplified cases (70% vs. 75%; log-rank p=0.86), but with a trend to lower 2-year OS in amplified cases (86% vs. 94%; log-rank p=0.28). Common grade 3 or 4 adverse events included constipation (0% vs. 8% of patients), neutropenia (12% vs. 4% of patients), anaemia (4% vs. 6% of patients), back pain (4% vs. 6% of patients), neuralgia (0% vs. 6% of patients) and neuropathy (8% vs. 4%).

Conclusions:

Four cycles of PAD induced high response rates in both 1q amplified and non-amplified myeloma with acceptable toxicity. After 2 years, EFS was also similar, but with a non-significant trend to lower OS in patients with 1q amplification.

Funding:

This study is sponsored by Janssen-Cilag Pty Ltd, Australia.

Trial Registration:

ACTRN12609000296235.

Disclosures:

Joshua: Janssen-Cilag Pty Ltd: Research Funding. Hertzberg:Janssen-Cilag Pty Ltd: Research Funding. Auguston:Janssen-Cilag Pty Ltd: Research Funding. Spencer:Novartis: Honoraria; Janssen: Honoraria, Speakers Bureau; Celgene: Honoraria. Horvath:Janssen-Cilag Pty Ltd: Research Funding. Bashford:Janssen-Cilag Pty Ltd: Research Funding. Campbell:Janssen-Cilag Pty Ltd: Research Funding. Ashurst:Janssen-Cilag Pty Ltd: Employment. Wade:Janssen-Cilag Pty Ltd: Employment. Copeman:Janssen-Cilag Pty Ltd: Employment. Butcher:Janssen-Cilag Pty Ltd: Consultancy. Prince:Janssen-Cilag Pty Ltd: Research Funding.
Induction of T Cell Immunity Using a Multipeptide Cocktail Containing XBP1, CD138 and CS1 Peptides in Smoldering Multiple Myeloma
Authors: Bae Jooeun , Prabhala Rao H. , Song Weihua , Tai Yu-Tzu , Anderson Kenneth C. , Munshi Nikhil C
Published: October 2019

Abstract 5039

Smoldering multiple myeloma (SMM) patients are at high risk for progression to active multiple myeloma (MM), making them candidates for novel immunotherapeutic strategies to prevent or delay disease progression. Among potential strategies, the ability to induce cytotoxic T lymphocytes (CTL) against multiple immunogenic epitopes provides a framework for overcoming major therapeutic challenges including heterogeneity of tumor associated antigen expression, frequent mutations of specific antigens, and variability of the human T-cell repertoire among individuals. In this study, we provide evidence that a cocktail of four immunogenic HLA-A2 specific peptides, heteroclitic XBP1 US184–192, heteroclitic XBP1 SP367–375, native CD138260–268 and native CS1239–247, induces specific CTL response in T cells from SMM patients. Following repeated rounds of multipeptide stimulation, we induced development of CD8+ CTL from SMM patients' T cells. The multipeptide specific-CTL demonstrated polyfunctional immune activities including high levels of IFN-g production, cell proliferation and cytotoxicity against MM cells in an HLA-A2 restricted manner. The multipeptide-specific CTL displayed increased memory (CD45RO+) and activated (CD69+) CD3+CD8+ T lymphocytes, suggesting that a multipeptide vaccine has the potential to induce durable memory by generating specific memory CTL with characteristics of effector T cells against MM cells. In addition, the multipeptide-specific CTL demonstrated peptide-specific responses to each of the relevant epitopes including heteroclitic XBP1 US184–192, heteroclitic XBP1 SP367–375, native CD138260–268 and native CS1239–247, but not against an irrelevant HLA-A2-specific MAGE-3271–279 peptide in various functional assays including antigen-triggered CD137 (4-1BB) expression, IFN-g production and CD107a up-regulation. Therefore, these results suggest the potential of inducing a broad spectrum of immune responses against selected XBP1 unspliced, XBP1 spliced, CD138 and CS1 target antigens in SMM using multipeptide vaccination. In conclusion, these studies provide the framework for clinical trials of vaccination in patients with SMM to delay or prevent progression to active MM.

Disclosures:

Bae: Oncopep Inc. : Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Anderson:Oncopep Inc. : Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Munshi:Oncopep Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.
Anti-ABCG2 Monoclonal Antibody in Combination with Paclitaxel-Nanoparticles Against Cancer Stem-Like Cell Activity in Multiple Myeloma
Authors: Yang Cuiping , Dou Jun , Chen Baoan , Sun Yunyu , Wang Yonglu , Li Xueming , Zhu Han , Li Zhi
Published: October 2019

Abstract 5044

In spite of the past efforts and progress made in treatment multiple myeloma (MM), most MM patients have eventually relapsed and died of the cancer. Cancer stem cells (CSCs) are considered responsible for continued growth and recurrence of cancer. The purpose of this study was to investigate the effects of anti-ABCG2 monoclonal antibody (mAb) in combination with paclitaxel-Fe3O4 nanoparticles (PTX-NPs) on CD138−CD34−MM cancer stem-like cells (MM CSCs) isolated from MM cell line JJN3. In our results, the MM CSCs expressed higher levels of the ABCG2 transporter, exhibited high proliferative, clonogenic and migratory potency, and demonstrated strong drug resistance and tumorigenicity when compared to non-CD138−CD34−MM cells. Incubation of mAb with PTX-NPs remarkably induced G/M cell cycle arrest, and increased synergistic induction of MM CSC apoptosis compared with incubation with PTX-NPs or PTX or mAb alone. More importantly, mAb in combination with PTX-NPs led to a significant reduction in the tumor volume, to a visible alleviation of murine lytic bone lesions and to a markedly increased survival rate in contrast to using a single agent in MM CSCs when it was transplanted to nonobese diabetic/severe combined immunodeficiency mice. Our study is the first to report on the anti-MM CSC activities by PTX-NPs as single agent or used together with mAb to treat MM. This finding from our study provides a rationale for future clinical trials.

Disclosures:

No relevant conflicts of interest to declare.
Nitrite Inhibits Platelet Reactivity in Presence of Deoxygenated Red Blood Cells As Measured by Flow Cytometry
Authors: Akrawinthawong Krittapoom , Piknova Barbora , Sibmooh Nathawut , Fucharoen Suthat , Schechter Alan N.
Published: October 2019

Abstract 5146

Nitric oxide (NO), a small gas molecule with multiple functions in the body, has been shown to have several precursors in body, the most abundant of which is nitrite. In the last decade several studies show that nitrite is reduced to NO by various enzymatic and non-enzymatic reductive processes, especially at low oxygenation conditions. These pathways are alternative mammalian sources of NO as compared to its enzymatic production by a family of nitric oxide synthase enzymes (NOS). NO has long been known to be a potent inhibitor of platelet function but the physiological and pathological implications of this has not been well clarified. We recently showed that addition of nitrite into plasma in the presence of red blood cells inhibits platelet aggregation and that this effect, as measured by cGMP production is enhanced by deoxygenation of erythrocytes. In order to study these effects more thoroughly on the molecular level, we have been developing flow-cytometric assays to study molecular processes on the platelet cell membrane known to correlate with platelet aggregation. We focused on P-selectin (CD62P) and activated GpIIb/IIIa (PAC-1) levels in platelets exposed to three activators, ADP, collagen and thrombin, in the presence of various concentrations of red blood cells and at variable levels of oxygen. We found that nitrite at physiological concentration (0. 1 to 1. 0 uM) significantly inhibited exposure of both of these surface markers on the outer membrane of platelets at levels of hematocrit above 23% and mean oxygen levels lower than 49 mmHg. The effect increased with increasing hematocrit and decreasing oxygen saturation into the hypoxic range. Addition of an NO scavenger prevented nitrite-related platelet inhibition, but addition of an inhibitor of NOS or of xanthine oxidoreductase had no effect. These results indicate that physiological levels of nitrite ions in the blood can modulate platelet reactivity in the presence of partially deoxygenated erythrocytes by the reduction to NO and that this process can modulate platelet activation and aggregation levels and confirm and extend our previous report (Srihirun et al, PLoS One 7, e30380, 2012), done primarily using aggregation assays. Our flow cytometry assays not only allows a more detailed and systematic study of the kinetics and equilibrium aspects of the aggregation process, but also offers a possibility to identify the individual molecular processes involved in these effects. The results may also explain certain differences between arterial and venous thrombosis.

Disclosures:

Schechter: NIH: Employment, co-inventor of NIH patent on use of nitrite in disease, co-inventor of NIH patent on use of nitrite in disease Patents & Royalties.
SNX3 Regulates Recycling of the Transferrin Receptor and Iron Assimilation in Developing Erythrocytes*
Authors: Chen Caiyong , Santos Daniel Garcia , Ishikawa Yuichi , Seguin Alexandra , Li Liangtao , Fegan Katherine H. , Hildick-Smith Gordon J. , Cooney Jeffrey D. , Chen Wen , King Matthew J. , Schultz Iman J. , Yien Yvette Y. , Dalton Arthur J. , Kingsley Paul D , Palis James , Hattangadi Shilpa , Lodish Harvey F. , Ward Diane M. , Kaplan Jerry , Maeda Takahiro , Ponka Prem , Paw Barry H.
Published: October 2019

Abstract 607

Developing erythrocytes acquire large amounts of iron through the transferrin (Tf) cycle for heme synthesis. The Tf cycle involves unidirectional transport of transferrin-transferrin receptor 1 (Tf-TfR1) complexes from the plasma membrane to the early and recycling endosomes (Figure). Besides the requirement for the basic trafficking machinery, specific sorting molecules exist to ensure the efficient re-cycling of Tf-TfR1 complexes and targeted iron delivery. The trafficking of TfR1 from recycling endosomes to the cell surface was shown to be mediated by Sec15L1, an exocyst component, as its mutation causes anemia in the hemoglobin deficit (hbd) mouse. The sorting mechanisms responsible for earlier trafficking steps in intracellular transferrin cycle, however, are poorly understood.

Here we report that sorting nexin 3 (SNX3), a cargo-specific retromer component, facilitates the endocytic recycling of TfR1, and thus, is required for the proper delivery of iron to erythroid progenitors for heme synthesis (Figure). Snx3 is highly expressed in hematopoietic tissues of zebrafish and mouse. Morpholino-mediated knockdown of snx3 in zebrafish embryos leads to a profound anemia. shRNA silencing of Snx3 in mouse primary fetal liver cells and mouse Friend erythroleukemia (MEL) cells inhibits the production of hemoglobin. We demonstrate that these defects are due to impaired transferrin-mediated iron uptake and delivery to the mitochondria. The impaired iron assimilation can be complemented with non-transferrin bound iron chelates, such as Fe-SIH (salicylaldehyde isonicotinoyl hydrazone). Furthermore, we show that SNX3 may act through direct physical interaction with TfR1 to sort Tf-TfR1 complexes to the recycling endosomes.

Our data from genetic, biochemical, and chemical biological studies collectively show that SNX3 regulates TfR1 trafficking and iron homeostasis in developing erythrocytes. The identification of SNX3 as an essential co-regulatory protein that regulates Tf-mediated iron delivery for heme synthesis provides a new genetic tool for exploring human disorders of iron metabolism, such as the hypochromic anemias, and erythropoiesis.

* Cell Metabolism (in revision).

Disclosures:

No relevant conflicts of interest to declare.
Coordinating Self-Renewal and Proliferation via MLL1-HOX Pathways and Beyond
Authors: Ernst Patricia , Artinger Erika L. , Mishra Bibhu P. , Zaffuto Kristin M. , Li Bin E. , Chen Yufei
Published: October 2019

Abstract SCI-28

Epigenetic regulation of gene expression plays a central role in normal hematopoietic stem cell (HSC) maintenance and leukemogenesis. The histone methyltransferase, MLL1, is essential for the maintenance of HSCs and is a common target of chromosomal translocations that result in acute leukemia. To discover genetic networks regulated by MLL1 in HSCs, we identified genes that were acutely deregulated upon Mll1 loss in HSCs, using a conditional knockout approach and lineage-negative, c-Kit+, Sca-1+, CD48-negative (LSK/CD48neg) cells. The majority of genes that changed were proliferation-associated genes, upregulated in Mll1−/− LSK/CD48neg cells. This reflected the fact that Mll1-deficient HSCs exhibit increased proliferation in vivo, a phenotype previously documented using the Mx1-cre inducible model. To determine whether the increased proliferation was cell-intrinsic, we performed single cell proliferation studies in serum-free medium containing SCF, IL-11, and Flt3L. We found that Mll1−/− LSK/CD48neg single cells entered the cell cycle earlier and that each cell cycle was shorter than wild-type controls. Evidence for failure to suppress lineage-specific gene expression was also observed; specifically, five percent of the upregulated genes encoded erythroid-specific proteins. These included erythroid transcriptional regulators such as GATA1 and KLF1, but also structural proteins such as spectrin, KEL, and EpoR. The relationship between erythroid-lineage genes and Mll1 was unique, since no other lineage-specific programs were upregulated in Mll1−/− LSK/CD48neg cells. Among the genes downregulated upon Mll1 loss, the largest category was comprised of transcriptional regulators, including Mecom, Pbx1, and Prdm16, which are known to control HSC self-renewal and quiescence. As observed in many other tissues, Mll1−/− LSK/CD48neg cells also exhibited reduced Hoxa9 expression. Interestingly, not all identified MLL1 target genes required menin, a cofactor thought to participate in directing MLL1 to particular genomic loci in vivo, and not all targets were Mll1-dependent in nonhematopoietic tissues. Chromatin immunoprecipitation and functional studies suggest that the identified genes act within a series of parallel pathways as direct transcriptional targets of MLL1. Interestingly, reexpression of Prdm16 alone could rescue Mll1-deficient cells from rapid attrition in bone marrow chimeras. Furthermore, Prdm16 corrected the hyperproliferation phenotype of Mll1−/− LSK/CD48neg cells. These data demonstrate that MLL1 coordinately regulates proliferation, lineage-specific gene expression programs, and self-renewal. By elucidating the normal MLL1-dependent transcriptional network within HSCs, we show that this pathway is overlapping but distinguishable from the leukemogenic pathway, suggesting that targeted therapy with minimal side effects on hematopoiesis will be feasible.

Disclosures:

No relevant conflicts of interest to declare.
The Impact of Novel Molecular Markers on Risk Stratification in Acute Myeloid Leukemia
Authors: Löwenberg Bob , Valk Peter , Touw Ivo P. , Delwel Ruud
Published: October 2019

Abstract SCI-33

The treatment of acute myeloid leukemia (AML) remains associated with considerable failure rates. From a therapy viewpoint AML presents as a kaleidoscope of highly diverse diseases for which the (epi)genetic heterogeneity of the disease is the overriding determinant of treatment failure. Diverse coemerging molecular abnormalities (gene mutations, expression abnormalities) related, for instance, to the genes CEBPA, nucleophosmin-1, FLT-3, RUNX1, ASXL1, P53, G-CSF-R, IDH1/IDH2, DNMT3A, and EVI-1 in variable composite configurations, determine the considerable “interindividual” and “intraindividual” variations of AML. As these somatic genetic abnormalities in transformed hematopoietic progenitor cells perturb critical cellular pathways and functions, they confer a profound impact upon the clinical phenotype of the disease—and also may define unique diagnostic subtypes (diagnosis) and therapeutic resistance (treatment response). Using genome-wide approaches and well-defined cohorts, the identified molecular biomarkers provide insights into the variability of treatment outcome, and furnish an informative framework for risk-adapted treatment decisions according to disease risk and therapeutic responsiveness (prognosis and treatment choice). In this presentation, examples of strategies to assess the prognostic impact of genomic abnormalities will be discussed, as well as the challenge to integrate the numerous markers into meaningful decision algorithms that can be used for more individualized therapy choices, for example, regarding the use of particular therapeutic agents and the utility (risk-benefit) of allogeneic stem cell transplantation.

Disclosures:

Löwenberg: Skyline Diagnostics: Membership on an entity's Board of Directors or advisory committees.
Gut Microbiota and Intestinal Inflammation
Authors: Garrett Wendy S.
Published: October 2019

Abstract SCI-49

Numerous studies of the microbiota that are found throughout the human body are under way, with the goal of unraveling the role of microbes in human physiology. Using experimental colitis models, we are investigating microbes that may instigate chronic inflammation, and we are studying putative beneficial microbes whose reduced presence may impact not only host response to the microbiota but also the behavior of the endogenous microbiota. Genomic approaches, combined with following microbial community response to a spectrum of dietary and pharmacologic perturbations, are shedding light on the dynamic operations of the microbiota that influence health and disease. Chronic inflammation in the intestine is not only the central pathophysiologic mechanism of inflammatory bowel disease (IBD) but also a key contributor to colorectal cancer. Ongoing work on the colorectal microbiome, using experimental models and human tumors, will be discussed. Collectively, our studies support the utility of wedding culture-independent and culture-dependent studies with mouse models for defining how the gut microbiota works in concert with the mucosal immune system to shape disease susceptibility for IBD and colorectal cancer.

Disclosures:

Garrett: Groupe Danone: Research Funding.
Dual SYK/JAK Inhibition Has a Broader Anti-Tumor Activity In Both ABC and GCB Types Of Diffuse Large B Cell Lymphoma
Authors: Ma Jiao , Coffey Greg , Lu Pin , Cheng Shuhua , Guo Ailin , Pandey Anjali , Wang Y. Lynn
Published: October 2019

With increasing understanding of the genetic basis of diffuse large B cell lymphoma (DLBCL), more targeted therapies have been developed. However, most of these therapies have activities only against a subset of DLBCL, activated B-cell like subtype (ABC), in particular, where the BCR pathway is known to be chronically active. Specific single-molecule directed therapies, although effective, often induce resistance over long term treatment. Agents with broader activities are needed for the treatment of this heterogeneous disease. Since both the BCR and JAK/STAT pathways are strongly implicated in the pathogenesis of DLBCL, in the present study, we evaluated the anti-lymphoma activity of PRT062070 (PRT2070), a novel compound that dually targets both JAK and SYK signaling pathways. We analyzed a panel of DLBCL cells lines, of both ABC and germinal center B-cell like (GCB) subtypes, as well as DLBCL patient samples, for cellular and molecular events affected by PRT2070.

Immunoblotting analyses showed that ABC and GCB subtype of DLBCL cells exhibit different JAK/STAT and BCR signaling profiles. For instance, AKT was highly expressed in GCB cells, whereas STAT3 was more strongly expressed in ABC cells. In GCB cell lines, PRT2070 blocked G1/S transition and caused cell cycle arrest. In contrast, in ABC cells, the drug induced apoptosis and down-regulated MCL1 protein. Furthermore, array of PRT2070-treated ABC subtype of DLBCL cells revealed that several STAT3 regulated cytokines and chemokines, including IL10, were down-regulated, confirming that PRT2070 affects ABC-DLBCL cells via JAK/STAT pathway. Genetic knockdown of both JAK and SYK reduced the growth and the survival of DLBCL cells more pronouncedly than knockdown of JAK or SYK alone, suggesting the anti-tumor activity of PRT2070 was mediated by dual inhibition of JAK and SYK signaling pathways. Importantly, JAK/STAT and BCR signaling can be blocked by PRT2070 in GCB and non-GCB primary human DLBCL cells, which led to death of these cells. Our work provided mechanistic insights into the actions of JAK/SYK dual inhibitor PRT2070 suggesting that the drug may be a potent treatment of DLBCL with a broader anti-tumor activity in both ABC and GCB subtypes of the lymphoma.

Disclosures:

Coffey: Portola Pharmaceuticals: Employment. Pandey:Portola Pharmaceuticals: Employment.
Mir-15a/16-1 Haploinsufficiency Contributes To Myeloma Progression
Authors: Chesi Marta , Garbitt Victoria , Palmer Stephen , Bergsagel P. Leif
Published: October 2019

Deletions of chromosome 13 occur in about 50% of multiple myeloma (MM) patients, with monosomy being the most common (85%), and small interstitial deletions, often involving the Rb gene, representing only 15% of cases. In contrast to CLL, where a minimal deleted region (MDR) has been mapped to the DLEU2/miR-15a/16-1 locus, no MDR has been identified in MM. The prognostic significance of monosomy 13 has been overshadowed by its strong association with high-risk MM, mostly t(4;14) and t(14;16) translocations. Genomic analysis of 28 MM tumors from Vk*MYC mice identified recurrent (27%) monosomy of the syntenic chromosome 14, with focal bi-allelic deletions of Rb1 in three independent tumors. One tumor presented a focal monoallelic interstitial deletion of the mirR15a-16.1 cluster. Reasoning that the biological significance of monosomy 13 would be conserved between human and murine MM, we carried out genetic crosses between Vk*MYC and mice heterozygous for Rb1 (MYC/Rb1), for the DLEU2/miR-15a/16-1 locus (MYC/MDR) or for the miR-15a/16-1 cluster only (MYC/MIR).

All of the MYC/Rb1 mice died around one year of age of pituitary tumors without a significant plasma cell expansion compared to the Vk*MYC mice alone. In contrast, both the MYC/MDR+/- and MYC/MIR+/- mice exhibited a progressive monoclonal plasmacytosis affecting secondary lymphoid organs, with associated shorter overall survival. The development of extra-medullary MM was further accelerated in MYC/MDR -/- and MYC/MIR -/- mice, and was reminiscent of that observed in Vk*MYC crossed with Emu-BCL2 mice. As previously shown for the CLL-like disease that develops in the MIR and MDR mice, the MM phenotype is more pronounced in the MYC/MDR than the MYC/MIR, and in the homozygous than the heterozygous mice. Although BCL2 has been proposed as a target of the miR-15a/16-1, it has not been identified among genes whose expression is altered in MIR null B lymphocytes, where instead miR-15a/16-1 deletion promotes proliferation. In contrast, we found significant BCL2 upregulation in some MM tumors from MYC/MIR and MYC/MDR, suggesting that miR-15a/16-1 deletion in MM provides a survival signal that renders plasma cells less dependent on the BM microenvironment. We propose that selection for monosomy 13 in MM is driven in at least in part by loss of one copy of the miR cluster.

Disclosures:

No relevant conflicts of interest to declare.