bluuebunny/crossref_metadata_2025 huggingface dataset:
CD4 and CD8 TCRαβ Cells Are selected On MHC Expressed On Thymocyte Precursors in OP9-DL1 Cultures.
Authors: Van Coppernolle Stefanie , Vandekerckhove Bart , Verstichel Greet , Velghe Imke , Taghon Tom , Leclercq Georges , Plum Jean , Kerre Tessa C. C.
Published: October 2019

Abstract 3670

Poster Board III-606

Human CD34+ haematopoietic precursor cells cultured on delta-like-ligand 1 expressing OP9 (OP9-DL1) stromal cells differentiate to T lineage cells. The nature of the T cells generated in these cultures has not been studied in detail. Since these cultures do not contain thymic epithelial cells (TEC) which are the main cell type mediating positive selection in vivo, generation of conventional helper CD4+ and cytotoxic CD8+ TCRαβ cells is not expected.

Phenotypically mature CD27+ CD1− TCRγδ as well as TCRαβ cells were generated in OP9-DL1 cultures. CD8 and few mature CD4 single positive (SP) TCRαβ cells were observed. Mature CD8 SP cells consisted of two subpopulations: one expressing mainly CD8αβ and one expressing CD8αα dimers. TCRαβ CD8αα and TCRγδ cells both expressed the IL2Rβ receptor constitutively and proliferated on IL-15, a characteristic of unconventional T cells. CD8αβ+ and CD4+ TCRαβ+ cells were unresponsive to IL-15, but could be expanded upon TCR stimulation as mature CD8αβ+ and CD4+ T cells. These T cells had the characteristics of conventional T cells: CD4+ cells expressed ThPOK, CD40L and high levels of IL-2 and IL-4; CD8+ cells expressed EOMES, RUNX3 and high levels of granzyme, perforin and IFN-γ.

Induction of murine or human MHC class I expression on OP9-DL1 cells had no influence on the differentiation of mature CD8+ cells, whereas blocking MHC class I antibodies and CD8 antibodies inhibited generation of mature CD27+ CD8+ SP cells. The presence of dendritic cells was not required for the generation of mature CD4+ or CD8+ T cells.

In conclusion, we have demonstrated positive selection of TCRαβ+ cells in the absence of TEC. The data suggest that positive selection is induced by interaction between T precursor cells without requirement for other haematologic or stromal elements.

Disclosures:

No relevant conflicts of interest to declare.
Incidence of Progressive Multifocal Leukoencephalopathy in Non-Hodgkin Lymphoma Patients Treated with Rituximab.
Authors: Focosi Daniele , Tuccori Marco , Blandizzi Corrado , Del Tacca Mario , Petrini Mario
Published: October 2019

Abstract 3675

Poster Board III-611

Progressive multifocal leukoencephalopathy (PML) is a rare complication of immunesuppression due to productive reactivation of JC polyomavirus (JCV) in glial cells. In the last decades many case reports of rituximab-associated PML have raised concerns, but no systematic incidence rate analysis has been ever performed. We retrospectively reviewed our clinical records with the aim of calculating the incidence rate of PML in patients with NHL who had been treated with regimens including rituximab. Data on HIV-negative patients who received the first dose of rituximab from January 1, 2000 to June 30, 2008, were analyzed. The follow-up period was from the first rituximab dose to the last recorded visit, up to September 30, 2008. PML cases were included if symptoms occurred at least 1 month after the first dose of rituximab and the diagnosis was supported by magnetic resonance imaging and detection of JCV DNA in stereotactic brain biopsies and/or cerebrospinal fluid. We collected data from 821 consecutive patients throughout the follow-up period. All patients received chemotherapy other than rituximab, and all completed their entire treatment course at the Hematology Unit in Pisa. No radiation therapy was administered. The median time of follow-up was 20 months (range = 1-106 month), resulting in 1725 patient-years at risk. Five cases of PML (two receiving maintenance rituximab) were identified, with an incidence rate of 2.89 cases per 1000 patient-years. We found that the incidence rate of PML in our population exceeded that observed in patients who are traditionally regarded as being at high risk of PML, namely patients with B-cell chronic lymphocytic leukaemia and AIDS. Rituximab might be a potential contributing factor to the development of PML in these patients. Nevertheless, rituximab currently represents an essential therapeutic tool that can positively affect the natural history of NHL. However, because of the expanding therapeutic indications, it seems reasonable to investigate the potential contribution of rituximab to PML occurrence.

Disclosures:

No relevant conflicts of interest to declare.
Latent Murine Herpesvirus-4 Infection Arms NK Cells.
Authors: White Douglas W , Keppel Catherine R , Schneider Stephanie E , Coder James , Reese Tiffany A , Payton Jacqueline E. , Ley Timothy J. , Virgin Herbert W , Fehniger Todd A
Published: October 2019

Abstract 3678

Poster Board III-614

Natural killer (NK) cells are lymphocytes originally identified by their ability to kill target cells without prior sensitization. However, murine NK cells require an antecedent ‘arming’ event to translate cytotoxic effector proteins (perforin and granzymes) resulting in potent cytotoxic capacity. In contrast to mice, most NK cells (CD56dim) from the peripheral blood of healthy humans are armed with pre-formed perforin and granzyme B proteins and mediate killing of NK sensitive targets directly ex vivo. This suggests that NK cells from specific pathogen free (SPF) laboratory mice are lacking a critical arming event present in healthy humans, and provides an experimental system to elucidate the events that result in NK cell arming in vivo. Since latent herpesviruses are highly prevalent and alter multiple aspects of host immunity, we hypothesized that the immune environment created by a latent herpesvirus infection arms NK cells. NK cells from mice latently infected with Murid herpesvirus 4 (MuHV-4), a virus closely related to the human viruses Kaposi's sarcoma associated herpesvirus (KSHV) and Epstein-Barr virus (EBV), were armed as evidenced by increased granzyme B protein expression, cytotoxicity, and interferon-gamma production. Adoptive transfer experiments with naïve NK cells indicated that NK arming occurred rapidly (≤ 72 hours) in the latently infected host and did not require acute infection. In addition, experiments utilizing a genetic variant of MuHV-4 that acutely infects mice similar to wild-type MuHV-4, but does not establish latency, confirmed that acute infection was not responsible for this NK cell modulation. Finally, NK cells armed by latent infection protected the host against a lethal RMA-S lymphoma challenge. Thus, the immune environment created by a latent herpesvirus infection arms NK cell function in vivo. These results indicate that some of the differences between human and SPF laboratory-mouse NK cell function may reflect a disparity in immune activation induced by chronic virus infection, as opposed to an intrinsic difference in the NK cells themselves. Moreover, these findings indicate that the activation state of the innate immune system needs to be taken into account when using mice to model immune responses against pathogens and tumors.

Disclosures:

No relevant conflicts of interest to declare.
Strong Inhibition of Alloreaction by Newly Generated Tolerogenic Dendritic Cells by Psoralen Plus UVA Treatment.
Authors: Maeba Hideaki , Nishimura Ryosei , Kuroda Rie , Araki Raita , Mase Shintaro , Ikawa Yasuhiro , Yachie Akihiro , Koizumi Shoichi
Published: October 2019

Abstract 3686

Poster Board III-622

Dendritic cells (DCs) are a heterogenous population of antigen-presenting cells (APCs) that contribute to innate immunity and initiate the adaptive immune response. In addition, recent studies demonstrated the existence of tolerogenic DCs (TDCs) that suppress the immunoreaction. Although the tolerogenic mechanisms are not fully understood, there are some methods reported to generate TDCs from conventional DCs by using cytokines such as IL-10/TGF-beta, vasoactive intestinal peptide (VIP), and IL-21. With the purpose of potential application of TDCs in a clinical bone marrow transplantation for preventing graft-versus-host disease (GVHD), which is caused by strong immunoreaction between host-typed conventional DCs and donor-typed naïve T cells, the establishment of much safer and easier, and more efficient TDCs culture system would be needed. To this end, we investigated whether treatment of conventional DCs with psoralen plus UVA (PUVA), which is widely and safely available in the treatment of some human immune disease and organ transplantation for preventing graft rejection, induced a subset of highly potent TDCs. Bone marrow cells obtained from Balb/c (H-2d) or C57BL/6 (H-2b) were incubated in complete RPMI containing GM-CSF for 10 days to generate bone marrow derived DCs (BM-DCs). BM-DCs were cultured with Psoralen (200 ng/mL) for 30 miniutes and then exposed to UVA light (2J/cm2). After 24 hours UVA irradiation, PUVA-treated DCs were collected and used in all experiments. First, PUVA-treated or untreated DCs with irradiatin were used as stimulator for allogenic splenocytes in mixed leukocyte reactions (MLR). The immunostimulatory capacity of PUVA-treated DCs was significantly diminished compared to those of untreated DCs (p<0.01). The expression levels of CD80 and CD86 by FACS, both of which are costimulatory molecules for T cell activation, was significantly reduced after PUVA treatment (p<0.05). This might explain for the induction of hyporesponsiveness in part. Next we further evaluated whether PUVA-treated DCs directly suppress T cell alloreaciton by cell-to-cell contact. Proliferation was inhibited when PUVA-treated DCs from the stimulator strain were added to the coculture, with a maximum reduction in proliferation of 95% at a 1:1 or higher ratio of untreated DCs to PUVA-treated DCs (Attached file). In conclusion, PUVA-treated DCs directly inhibit T cell alloreaction. Infusion of host-typed PUVA-treated DCs would be potent strategy for preventing lethal acute GVHD.

Disclosures:

No relevant conflicts of interest to declare.
The mTOR Pathway Inhibits Expression of the Tumor Suppressors ELF4 and KLF4 Downstream of TCR Signaling to Induce T Cell Proliferation.
Authors: Yamada Takeshi , Gierach Kirsten , Lacorazza H. Daniel
Published: October 2019

Abstract 3688

Poster Board III-624

Quiescence of circulating naïve T cells is maintained by the transcription factors ELF4 and KLF4 downstream of T-cell receptor (TCR) signaling. Hence, loss of ELF4 leads to increased proliferation of CD8+ T cells in response to homeostatic and antigen driven stimuli (Yamada et al, Nature Immunology, 2009). The identification of signals that suppress this restraint of proliferation will aid to enhance immunological memory during vaccination and to better understand development of T-cell acute lymphoblastic leukemias. Consistent with lower threshold of activation by ELF4 deletion in unstimulated naïve T cells, we identified a significant downregulation of the dual-specificity phosphatases DUSP1 and DUSP5 in a global gene expression study, which was confirmed at a protein level. Consequently, Elf4−/− CD8 T cells showed sustained phosphorylation of Erk1/2 upon TCR activation. In addition, we found that the PD98059 and LY294002 inhibitors, but not Cyclosporin A, blocked inhibition of ELF4 transcription upon TCR activation independently of CD28 co-stimulation and signals emanating from IL-2R. Furthermore, rapamycin also prevented downregulation of ELF4 transcripts following T cell activation, suggesting that mTORC1 inhibits ELF4 transcription downstream of MAPK and PI3K/Akt pathways. We conclude that the transcription factor ELF4 sets a proliferation threshold in naïve T cells by activating DUSPs and that ELF4 suppression upon TCR activation is mediated by mTORC1 downstream of MAPK and PI3K/Akt pathways. Our findings provide important targets of this novel control of T cell proliferation to enhance immune response to vaccination and to prevent expansion of pre-leukemic clones in pediatric patients that fail to respond to current therapies.

Disclosures:

No relevant conflicts of interest to declare.
SNX 2112, An Oral Hsp-90 Inhibitor Exerts Antiproliferative Effects in Combination with Bortezomib and Rituximab in Rituximab Resistant Non-Hodgkin's Lymphoma.
Authors: Reddy Nishitha , Hicks Donna , Jagasia Madan , Amiri Kathy
Published: October 2019

Abstract 3733

Poster Board III-669

Background

Heat Shock protein 90 (Hsp90) is a chaperone that plays an important role in maintaining the stability of various other client proteins involved in cell survival and proliferation. Few client proteins that are dependent on Hsp-90 include Akt, Raf, ERK and NF-κB. Hsp-90 is upregulated in several malignancies and thus serves as an important therapeutic target. SNX 2112 (PF-04928473) is an oral heat shock protein inhibitor that is currently under phase-I clinical trials.

Methods and Results

We examined the effect of increasing concentrations of SNX 2112 in combination with other active targeted agents in Non-Hodgkin's lymphoma. In our current work, we exposed rituximab sensitive (RSCL) and rituximab resistant cell lines (RRCL) to varying concentrations of SNX-2112 (500nM, 1μM, μM, 10μM and 20 μM) along with fixed doses of rituximab (10μg/ml) as well as bortezomib (500nM) and performed growth inhibition assays. The growth inhibitory effects of the various agents in Lymphoma cell lines were assessed by measuring 3-2, 5-diphenyl tetrasodium bromide (MTT) dye absorbance. Cells were incubated for 24 hrs and 48hrs. MTT was added to each well the last 4 hrs. Absorbance was measured using spectrophotometer. Accordingly, immunoblotting was performed using lymphoma cell lysates. The whole cell lysates were subjected to polyacrylamide gel electrophoresis and nitrocellulose membranes were immunoblotted with phosphorylated Hsp-90 and α-tubulin. In parental raji cell lines, at 48 hrs, and a dose of 10 μM, SNX 2112 demonstrated potent anti-proliferative effects when combined with bortezomib and rituximab 0.23 OD vs. rituximab alone 1.06 OD (p=0.010) 95% CI; 0.47, 1.18. In rituximab resistant cell lines, SNX 2112 with rituximab and bortezomib demonstrated significant antiproliferative effects 0.199 OD vs. rituximab alone 0.86 OD (p=0.023) 95% CI 0.22-1.11. As a single agent neither rituximab or bortezomib nor the combination of these agents was significant in inhibiting cell growth in rituximab resistant cell lines. Cytotoxicity was more significant in the rituximab resistant cell lines that rituximab sensitive raji cell lines. Immunoblotting performed using phosphorylated hsp-90 also demonstrated significant inhibition when treated with SNX 2112.

Conclusions

Our current research demonstrates that SNX 2112 when added to bortezomib and rituximab has augmented anti-tumor activity (ie, antiproliferation) and demonstrates additive effects. Further in vivo studies are underway to demonstrate this effect. Our promising pre-clinical results of this unique combination support further evaluation in a clinical trial in patients with relapsed Non-Hodgkin's lymphoma.

Disclosures:

No relevant conflicts of interest to declare.
Treatment Model of CNS Lymphoma Using Complement-Dependent Cytotoxicity Induced by Rituximab and Autoserum.
Authors: Miyake Yasuyuki , Okoshi Yasushi , Machino Takayuki , Chiba Shigeru
Published: October 2019

Abstract 3739

Poster Board III-675

Background

Primary central nervous system lymphoma (PCNSL) is almost exclusively CD20-positive non-Hodgkin lymphoma (NHL). Although rituximab (R) is widely used for CD20-positive NHL, it is not considered to reach brain lesions effectively beyond the blood brain barrier. Intraventricule administration (ivt) of R is reported to be effective in meningeal lymphoma but the effect on lesions in the brain parenchyma seems to be limited. Recently, a case of refractory PCNSL that was successfully treated with ivt of R with autologous serum was reported (Takami A, et al. Cancer Science, 2006). Because the cerebrospinal fluid does not contain complements which exists in the serum, induction of complement-dependent cytotoxicity by ivt of R plus autoserum was speculated. To investigate this effect, we developed an animal treatment model of CNSL.

Materials and methods

Raji, CD20-positive Burkitt lymphoma cell line, was inoculated into the deep frontal lobe of the brain of 8-week old F344 (nru-/nru-) nude rats, using brain stereotaxic apparatus. At the same time, a cannula was placed into the ipsilateral lateral ventricle. After several days, R or control immunoglobulin (cIg), plus human serum or saline, was administrated into the lateral ventricle.

Results

The brain was extracted 24 hours after the last administration and frozen section was made. Human CD20-positive Raji cell tumor was also positively stained with FITC-conjugated anti-human IgG antibody when R but not cIg was administrated. Consequently, R in the lateral ventricle was considered to penetrate ependymal cells and brain parenchyma, and bound to lymphoma cells. Next, these rats were treated with ivt of R plus serum (R + Serum), cIg plus serum (cIg + Serum), or R plus saline (R + saline). These were administrated once a day from day 5 to day 9 after inoculation of Raji, and then survival was monitored. When an obvious weakness, such as marked and consecutively loss of activity or weight, was observed, these rats were euthanized and this is defined as dead day. In each case, the brain was extirpated and examined whether lymphoma existed or not. Death without lymphoma or from technical problem was excluded from the analysis. Survival of each group was analyzed by Kaplan-Meier method and log-lank test. R + Serum group had longer survival than cIg + Serum (p = 0.049). Long-term survivors were only seen in R + Serum and this group seemed to be superior to R + saline but statistical difference was not detected (p = 0.083). There were no difference between cIg + Serum and R + saline (p =0.382) and neither group had long-term survivor.

Conclusion

The possibility of novel treatment of CNSL with ivt of R and autoserum was shown in the rat CNSL model. To confirm this approach, clinical trials are warranted.

Disclosures:

No relevant conflicts of interest to declare.
A Phase II Trial of R-FM (Rituximab, Fludarabine and Mitoxantrone) Chemotherapy Followed by Yttrium 90 (90Y) Ibritumomab Tiuxetan (90Y-IT) for Untreated Follicular Lymphoma (FL) Patients.
Authors: Zinzani Pier Luigi , Tani Monica , Broccoli Alessandro , Derenzini Enrico , Pulsoni Alessandro , De Renzo Amalia , Fanti Stefano , Fina Mariapaola , Gandolfi Letizia , Pellegrini Cinzia , Stefoni Vittorio , Baccarani Michele
Published: October 2019

Abstract 3743

Poster Board III-679

Introduction

In 2008 we published a multicenter non-randomized phase II trial of fludarabine and mitoxantrone plus 90Y-IT in untreated patients with FL. By the end of the entire treatment regimen 95% of the patients achieved complete remission (CR). With a median follow-up of 30 months, 3-year PFS was estimated to be 76% and 3-year OS 100%. On the basis of these results we are currently conducting a prospective, multicenter, non-randomized, phase II study of R-FM followed by 90Y-IT in untreated patients with FL in which the number of fludarabine and mitoxantrone cycles has been decreased to four and in which rituximab is administered before each cycle. The rationale of the trial is to use different forms of treatment and to reduce the use of conventional chemotherapy and its related toxic effects.

Patients and Methods

Patients eligibility is represented by: age more than 18, stage II-IV, FL grade I-II, WHO performance status 0-2. Patients are treated with standard FM chemotherapy plus rituximab every 28 days for 4 cycles. Patients are restaged 4 to 8 weeks after completion of immunochemotherapy and those achieving at least a partial response are eligible for 90Y-IT. All patients receive a single dose of 90Y-IT 14,8 MBq/kg. At the time of the analysis we enrolled 55 patients. 25 patients were male and 30 female; the median age was 56 years (range 26-84); 12 patients were stage II, 13 stage III and 30 stage IV; 11 patients had a bulky disease. 52 patients completed the induction chemotherapy, all except 5 were eligible for the consolidation treatment with 90Y-IT and 44 patients were restaged after the entire treatment regimen.

Results

After the R-FM chemotherapy, the overall response rate was 92.3% (48/52) including 39 (75%) CR and 9 (17.3%) partial remissions (PR). Time to event analyses, including TTP and duration of response are pending further follow-up. Treatment was well tolerated grade 3-4 haematologic AEs (mostly neutropenia) were seen in 50% of the patients. Among the 44 patients (9 PR and 35 CR) subsequentially treated with 90Y-IT and reassessed for the response, 8/9 (88.9%) PR patients improved their remission status from PR to CR. 90Y-IT toxicity included mostly grade 3-4 neutropenia and thrombocytopenia and was comparable to the literature data.

Conclusions

These preliminary data indicate that radioimmunotherapy appears highly effective and feasible as “consolidation” after short (4 cycles) immunochemotherapy in FL patients, improving quality of response without any cumulative toxicity.

Disclosures:

No relevant conflicts of interest to declare.
Ad-ISF35-Transduced Autologous Cells Promote in Vitro and In Vivo Chemosensitization to FCR in Patients with Del(17p) / P53 Defective Chronic Lymphocytic Leukemia.
Authors: Castro Januario E. , Melo-Cardenas Johanna , Sandoval-Sus Jose , Urquiza Mauricio , Prussak Charles , Kipps Thomas J.
Published: October 2019

Abstract 376

Chronic lymphocytic leukemia (CLL) cells that have del(17p) typically have loss of functional P53, rendering these cells refractory to standard chemotherapeutic agents, which require activation of P53 for their cytotoxic activity. However, del(17p) CLL cells co-cultured in vitro with cells transduced to express the CD40L (CD154) activate another member of the P53 family of proteins, namely P73, which, like P53, can induce transcription of death receptors and pro-apoptotic proteins and sensitize cells to the cytotoxic activity of “P53-dependent” drugs, such as Fludarabine (F-ara-A). Moreover, transduction of del(17p) CLL cells with a replication-defective adenovirus (Ad) encoding recombinant CD154 (Ad-ISF35) can induce such changes in both transduced and bystander CLL cells in vitro. To examine whether a similar activity could be obtained in vivo, we are conducting a phase Ib clinical study in subjects with high-risk CLL who are refractory to fludarabine or have evidence of del(17p). In this study, subjects receive three IV doses of 3×108 autologous CLL cells that had been transduced ex vivo with Ad-ISF35 and two weeks latter they are treated with a truncated chemoimmunotherapy regimen involving 3 monthly courses of fludarabine, cyclophosphamide and rituximab (FCR). P53-defective CLL cells from treated patients were initially resistant to F-ara-A induced apoptosis with IC50 > 10μM prior to treatment. CLL cells collected from patients ≥ 24 hours after the first infusion of autologous Ad ISF35-trasduced CLL cells became sensitive to the cytotoxic effects of F-ara-A, with IC50 0.3-1 μM. Enhanced sensitivity to F-ara-A was associated with induced expression of Bid, DR5, CD95, and P73 by circulating non-transduced “bystander” CLL cells, an effect lasting ≥ 2 weeks following IV infusion. To date, two subjects have completed treatment and this has been well tolerated without serious adverse events. The most common adverse events have been transient fever, malaise and fatigue associated to infusion of Ad-ISF35 transduced cells and cytopenias after treatment with FCR. Both subjects have achieved a compete response, one of them without detectable minimal residual disease (MRD) by sensitive multiparameter flow cytometry of marrow mononuclear cells obtained 3 months following completion of treatment. Moreover, these patients have complete resolution of lymphadenopathy and organomegaly by exam and by whole body computed tomography. These results indicate that Ad-ISF35-cell-gene therapy can sensitize P53-deficient CLL to “P53-dependent” cytotoxic agents in vivo, allowing for effective treatment of patients who otherwise would be resistant to standard forms of therapy.

Disclosures:

Prussak: Memgen:

Employment.
OSU-DY7, a Novel D-Tyrosinol Derivative, Mediates Cytotoxicity in Chronic Lymphocytic Leukemia and Lymphoblastic Lymphoma through p38 Mitogen-Activated Protein Kinase Pathway.
Authors: Bai Li-Yuan , Ma Yihui , Kulp Samuel K , Wang Shu-Huei , Chiu Chang-Fang , Frissora Frank , Mo Xiaokui , Jarjoura David , Byrd John C. , Chen Ching-Shih , Muthusamy Natarajan
Published: October 2019

Abstract 3778

Poster Board III-714

Introduction

Chronic lymphocytic leukemia (CLL) is a most common form of adult leukemia with minimal treatment options. Drug resistance and associated immune deregulation limit use of current therapies, thus warranting need for alternative therapy development. Our laboratories recently identified a novel D-tyrosinol-derived compound targeting p38 MAPK pathway, OSU-DY7 [(R)-2-amino-3-(4-heptyloxy-phenyl)-propan-1-ol]. Here we demonstrate the efficacy of OSU-DY7 for lymphocytic cell lines and primary B cells from CLL patients.

Materials and Methods

We tested the pre-clinical efficacy of OSU-DY7 in primary CLL B cells and B cell lines representing CLL (MEC-1), ALL (697), and lymphoblastic lymphoma (Raji and Ramos).

Results

The cytotoxicity of OSU-DY7 was both dose- and time-dependent. The IC50 of OSU-DY7 at 24 hrs for MEC-1, 697, Raji, Ramos and primary B-CLL cells were 2.40 μM, 10.39 μM, 6.04 μM, 3.75 μM and 3.58 μM respectively. OSU-DY7 induced activation of caspase-3 and poly (adenocine diphosphate-ribose) polymerase (PARP) cleavage. Pancaspase inhibitor Z-VAD-FMK partially rescued OSU-DY7-induced cytotoxicity in CLL B cells and cell lines. Interestingly, OSU-DY7 mediated-cytotoxicity was associated with increased phosphorylation of p38 MAPK and its downstream target protein MAPKAPK2 in both lymphocytic cell lines and primary B-CLL cells. Compared with control group, the ratio of p-p38MAPK (Thr180Tyr182) versus p38MAPK in Raji cells treated with 2 μM, 4 μM and 8 μM of OSU-DY7 increased 2.2, 4.7 and 11 fold respectively (p<0.0001). Similar increase in p38MAPK phosphorylation was also observed in six CLL patient samples after treatment with OSU-DY7 2 μM, 4 μM and 8 μM for 24 hours (p=0.0004). Moreover, SB202190, a specific p38 MAPK inhibitor, decreased MAPKAPK2 protein level with concomitant rescue of the cells from the OSU-DY7 mediated-cytotoxicity. Thus pretreatment of Raji and primary CLL B cells with SB202190 reduced the OSU-DY7 induced cytotoxicity by 36.7% and 24.3% respectively by 24hrs (Raji:p<0.0001; primary CLL B cells: p<0.005 ). Furthermore, SB202190 rescued OSU-DY7 down regulated survivin protein by ∼2 fold (p<0.0001, n=3) and mRNA levels by 2.4 fold (95% CI: 1.56∼3.84 fold, p=0.0026, n=3) in Raji cells indicating a role for p38MAPK dependent regulation of survivin by OSU-DY7.

Conclusions

This study provides an evidence for a role of OSU-DY7 in p38 MAPK dependent activation and survivin down regulation associated with apoptosis in lymphocytic cells, thus warranting further development of this novel agent for alternative therapy for lymphocytic malignancies. [This work was supported by D. Warren Brown Foundation and Leukemia and Lymphoma Society]

Disclosures:

No relevant conflicts of interest to declare.
The PI3K Inhibitor GDC-0941 Synergizes with Standard of Care Therapies to Induce Growth Inhibition and Apoptosis of Multiple Myeloma Cells.
Authors: Munugalavadla Veerendra , Berry Leanne , Du Changchun , Mariathasan Sanjeev , Slaga Dion , Sun Laura , Chesi Marta , Bergsagel Peter Leif , Ebens Allen J.
Published: October 2019

Abstract 3788

Poster Board III-724

Multiple myeloma (MM) is a malignancy characterized by clonal expansion and accumulation of long-lived plasma cells within the bone marrow. Phosphatidylinositol 3' kinase (PI3K) -mediated signaling is frequently dysregulated in cancer and controls fundamental cellular functions such as cell migration, growth, survival and development of drug resistance in many cancers, including MM, and therefore represents an attractive therapeutic target. Here, we demonstrate in vitro, that a potent and selective pan-isoform PI3Kinhibitor, GDC-0941, modulates the expected pharmacodynamic markers, inhibits cell-cycle progression and induces apoptosis; overcomes resistance to apoptosis in MM cells conferred by IGF-1 and IL-6; and is additive or synergistic with current standard of care drugs including dexamethasone, melphalan, lenolidamide and bortezomib. In cell lines we find sensitivity to GDC-0941 is positively correlated with pathway activation as determined by phospho-AKT-specific flow-cytometry and Western-blot analysis. Preliminary results indicate apoptosis of MM cells is correlated with increased expression of the proapoptotic BH3-only protein BIM; mechanisms of increased apoptosis in MM will be further explored and an update presented. We further extend these in vitro findings to show that GDC-0941 has activity as a single agent in vivo and combines well with standard of care agents in several murine xenograft models to delay tumor progression and prolong survival. Our results suggest that GDC-0941 may combine well with existing therapies, providing a framework for the clinical use of this agent, and a rational approach to improving the efficacy of myeloma treatment.

Disclosures:

Munugalavadla: Genentech: Employment, Patents & Royalties. Berry:Genentech: Employment, Patents & Royalties. Du:Genentech, Inc.: Employment, Equity Ownership. Mariathasan:Genentech: Employment, Patents & Royalties. Slaga:Genentech: Employment, Patents & Royalties. Sun:Genentech Inc.: Employment. Chesi:Genentech, Inc.: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Merck: Research Funding. Bergsagel:Genentech: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Merck: Research Funding. Ebens:Genentech, Inc.: Employment, Equity Ownership, Patents & Royalties.
Patients with Del(5q) MDS Who Fail to Achieve Erythroid or Cytogenetic Remission After Treatment with Lenalidomide Have An Increased Risk for Clonal Evolution and AML Progression.
Authors: Göhring Gudrun , Giagounidis Aristoteles , Büsche Guntram , Kreipe Hans , Zimmermann Martin , Hellstrom-Lindberg Eva , Aul Carlo , Schlegelberger Brigitte
Published: October 2019

Abstract 3818

Poster Board III-754

Lenalidomide consistently induces transfusion independence and complete cytogenetic response in patients with low and intermediate risk myelodysplastic syndromes (MDS) with 5q deletion. Only limited information on long-term outcome of patients treated with this novel approach is currently available. We performed a long-term follow-up analysis of 42 patients with low or intermediate risk MDS and 5q deletion treated with lenalidomide. At a median follow-up of 40 months, 58% of the patients achieved an erythoid response and 48% a cytogenetic response. 36% of patients progressed into acute myeloid leukaemia (AML). Most of them (87%) acquired chromosome aberrations in addition to the 5q deletion, i.e. underwent clonal evolution during leukaemogenesis. There were no clinical, cytological or cytogenetic markers at study entry that allowed prediction of an increased risk of leukaemic transformation. However,erythroid and cytogenetic responders had a significantly decreased risk of progression to AML (p=0.001 and p=0.009, respectively) compared to non-responders. 3 and 5 years after study entry, the cumulative incidence of AML for patients with a cytogenetic response was 10% and 21%, respectively, and for patients without cytogenetic response, it was 46% and 60%. Patients with del(5q) MDS who fail to achieve erythroid or cytogenetic remission after treatment with lenalidomide have an increased risk for clonal evolution and AML progression. In refractory, or relapsing, patients, genetic instability and clonal evolution seem to be the driving forces of leukaemic transformation. Regular follow-up investigations of del(5q) MDS patients treated with lenalidomide may help identifying patients needing alternative treatment strategies.

Disclosures:

Göhring: Celgene Corp.: cytogenetic reference lab for Celgene MDS-004 study. Giagounidis:Novartis Pharma: Consultancy; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Glaxo SmithKline: Consultancy; Johnson&Johnson: Consultancy; Celgene Corp.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Büsche:Celgene Corp.: histology reference lab for MDS-004 study. Kreipe:Celgene Corp.: histology reference lab for MDS-004 study. Hellstrom-Lindberg:Celgene Corp.: Research Funding. Schlegelberger:Celgene Corp.: cytogenetic reference lab for Celgene MDS-004 study.
Interleukin-6 (IL-6) Enhances C-MYC Translation IN MULTIPLE MYELOMA (MM) CELLS: ROLE of IL-6-INDUCED EFFECTS On the C-MYC RNA-Binding PROTEIN, HNRNPA1.
Authors: Shi Yijiang , Gera Joseph , Lichtenstein Alan
Published: October 2019

Abstract 3839

Poster Board III-775

Our previous work (Cancer Research 68:10215, 2008) demonstrated that IL-6 enhanced c-myc protein expression in MM cells and function of the RNA-binding protein, hnRNPA1 (A1), was required. This occurred by way of enhanced cap-independent translation mediated via the internal ribosome entry site (IRES) in the 5'UTR of the myc RNA. IRES-dependent translation is the fail safe mechanism for protein expression when cap-dependent translation is suppressed by mTOR inhibition (with curtailed RNA cap-ribosome binding) and is especially important when an mRNA leader is relatively long and highly structured, such that scanning ribosomes are unlikely to efficiently initiate translation. The IRES directly recruits the ribosome to within close proximity to the start codon, bypassing the need for cap binding and ribosome scanning. HNRNPA1 (A1) is a documented IRES trans-acting factor (ITAF) for the myc IRES, facilitating translation but how its effects are enhanced by IL-6 is unknown. HNRNPA1 is a shuttling protein which binds the myc RNA in the nucleus and transports it to the cytoplasm. Initial experiments demonstrated that IL-6 stimulation of ANBL-6 and OCI-My5 MM cell lines, as well as several primary MM specimens, significantly increased the cytoplasmic localization of A1. To test if this enhanced cytoplasmic localization was critical, we stably expressed a dominant negative (DN) A1 gene that is incapable of nuclear-to-cytoplasmic shuttling and which also prevents endogenous hnRNPA1 shuttling. The DN also prevented A1-mediated transport of the shuttling protein, FUS. Expression of the DN in ANBL-6 cells prevented IL-6-induced effects on myc expression and on ANBL-6 cell growth. We also tested if IL-6 treatment affected A1 binding to the myc RNA by immunoprecipitating A1 and performing real time PCR on the immunoprecipitate for myc RNA levels. A significant increase in A1-myc RNA binding was confirmed. Mass spectroscopy demonstrated that IL-6 induced phosphorylation of A1 in its RNA-binding domain, which possibly mediated the enhanced binding. These results demonstrate that, by enhanced binding of the myc ITAF, hnRNPA1, to the myc IRES, and by enhanced transport of the complex to the translationally active cytoplasmic subcellular site, IL-6 may stimulate c-myc translation and subsequent MM cell growth.

Disclosures:

No relevant conflicts of interest to declare.
Polycythemia Vera with Multiple Clones Carrying Different Mutations (L611V, V617F, L611V/V617F) in Exon 14 of JAK2.
Authors: Cleyrat Cédric , Jelinek Jaroslav , Girodon François , Boissinot Marjorie , Ponge Thierry , Harousseau Jean-Luc , Issa Jean-Pierre , Hermouet Sylvie , Hermouet Sylvie
Published: October 2019

Abstract 3908

Poster Board III-844

Background

In more than 95% of cases, Polycythemia vera (PV) is characterized by the presence of the V617F mutation of JAK2 (JAK2-V617F). We report on three cases of V617F-positive PV with an additional mutation of JAK2 changing leucine 611 for a valine (L611V).

Design and Methods

We used allele-specific quantitative PCRs and pyro-sequencing to detect the L611V and V617F JAK2 mutants in genomic DNA and in cloned PCR products. The consequences of the different mutations on the function of JAK2 were investigated using transient expression in BaF-3/EpoR cells.

Results

We found equal proportions of L611V and V617F alleles in granulocyte DNA (patients Na249, 19%; Na382, 26%; Di362, 27%) and established the presence of alleles with L611V and V617F in cis (L611V/V617F). The double JAK2-L611V/V617F mutant represented 15% to 26% of JAK2 alleles in granulocyte DNA. JAK2-L611V/V617F was detected in 12% to 30% of erythroid colonies, always in a heterozygous fashion. Low levels of single mutant alleles were also found, in 0.5% of cloned PCR products (JAK2-L611V, patient Na249) and in 1.5% of genomic DNA and cloned PCR products (JAK2-V617F, patient Na249), indicating that mutations in JAK2 occurred more than once. Functionally, JAK2-L611V was found comparable to wild-type JAK2, whereas JAK2-L611V/V617F displayed greater constitutive tyrosine phosphorylation of JAK2 and AKT than wild-type JAK2 and JAK2-V617F. The double JAK2-L611V/V617F mutant was associated with a high hematocrit and normal counts of leukocytes and platelets. Patients Na382 and Di362, but not patient Na249, carried the 46/1 haplotype of chromosome 9p associated with a reported pre-disposition to mutations of JAK2 on the same allele.

Conclusion

PV patients can harbor multiple clones carrying different mutations of JAK2, which may or may not increase JAK2 activity. Functionally silent mutations, not leading to clonal expansion, may remain undetected. The double JAK2-L611V/V617F mutated clone was predominant whereas single L611V and V617F JAK2 mutants represented <2% of JAK2 alleles. Thus presence of the activating JAK2-V617F mutation does not ensure clonal expansion, implying that other factors promote or repress expansion of JAK2-mutated progenitors. Lastly, mutations of JAK2 can occur more than once in patients negative for the pre-disposing 46/1 haplotype.

Disclosures:

No relevant conflicts of interest to declare.
The Overexpression of Enhancer of Zeste Homologue 2 Is Associated with Tumor Proliferation and Aggressive Behavior in Adult T-Cell Leukemia/Lymphoma.
Authors: Sasaki Daisuke , Imaizumi Yoshitaka , Hasegawa Hiroo , Osaka Akemi , Tsuruda Kazuto , Tsukasaki Kunihiro , Iwanaga Masako , Miyazaki Yasushi , Yanagihara Katsunori , Marquez Victor , Kamihira Shimeru , Yamada Yasuaki
Published: October 2019

Abstract 3959

Poster Board III-895

Background

Enhancer of zeste homologue 2 (EZH2) is a critical component of the Polycomb Repressive Complexes PRC2, which mediates epigenetic gene silencing by the trimethylation of Lys27 of histone 3 (H3K27). This regulation is not only involved in embryonic development and stem cell renewal, but also in tumor progression. Adult T-cell leukemia/lymphoma (ATLL) is a single disease entity etiologically associated with human T-cell leukemia virus type-1 (HTLV-1). Its clinical behavior, however, is quite diverse among patients and is thus subclassified into several subtypes. The prognosis of the aggressive subtypes is very poor based on standard chemotherapy, and so the development of a new therapeutic approach is urgent.

Results

In a comparative microarray analysis of primary ATLL samples, the acute type showed significantly higher EZH2, YY1, and RYBP (RING1 and YY1 binding protein) expressions compared with the chronic type. Further analysis employing real-time quantitative RT-PCR of various Polycomb group (PcG)-related proteins, including the ones mentioned above, revealed that the mRNAs of two other PRC2 components, EED and RBBP4, were also significantly elevated in ATLL, irrespective of the subtypes, compared with lymphocytes from HTLV-1 carriers. These results suggest that the dysregulation of PRC2 is a key event in the development and progression of ATLL. In the immunohistochemical analysis of ATLL lymph nodes, the nuclei of over 90% of ATLL cells were positive for EZH2, which in clear contrast to reactive or indolent B-lymphoma lymph nodes in which a few cells were positive. It has been shown that activated Akt phosphorylates EZH2 at serine 21 and suppresses its methyltransferase activity. Western blotting of EZH2 showed that EZH2 of primary ATLL cells was not phospholyrated and remained in its active form. Indeed, the trimethylation of H3K27 in ATLL cells was confirmed by Western blotting and immunohistochemistry, whereas it was completely absent in lymphocytes from HTLV-1 carriers. Finally, in studies using ATLL cell lines, the knockdown of EZH2 by siRNA caused decrease in cell proliferation. Moreover, ATLL cell lines showed high sensitivities to histone deacetylase (HDAC) inhibitors as LBH589, and LBH589 decreased EZH2 expression. EZH2 inhibitor 3-deazaneplancin A (DZNeP) also reduced cell growth, not only in primary ATLL cells, but also in ATLL cells lines. These results strongly support the rationale for developing molecular targeting therapies against EZH2 in ATLL.

Disclosures:

No relevant conflicts of interest to declare.
Oncogenic Pim Kinase Activity Provides Resistance to Mtor Inhibition in Vitro and In Vivo.
Authors: Schatz Jonathan H , Wendel Hans-Guido
Published: October 2019

Abstract 3974

Poster Board III-910

The PI3K/Akt/mTor pathway is among the most frequently deregulated in human cancers, including many leukemias and lymphomas, and inhibitors targeting it at multiple levels are either clinically available or under development. The Pim family proteins are oncogenic serine/threonine kinases expressed in many malignancies and that have numerous overlapping downstream targets and functional consequences with PI3K/Akt/mTor. We here investigate the role of Pim proteins in oncogenesis and their ability in particular to mediate resistance to mTor inhibition. We find over-expression and genomic amplification of Pim1 and Pim2 in tumor samples and cell lines derived from patients with multiple B lymphomas. We show that both Pim1 and Pim2 powerfully mediate resistance to apoptosis in the murine pro-B cell line FL5-12 when withdrawn from IL-3. In murine tumor cells with constitutive activation of mTor, Pim1 and Pim2 provide resistance to treatment with the mTor inhibitor rapamycin. In vivo, we find that murine Pim2 (mPim2) accelerates tumorigenesis in the Eu-Myc transgenic model of Burkitt's lymphoma in a manner highly similar to Akt. However, unlike Akt, mPim2 leads to tumors that resist sensitization to chemotherapy by co-administration of rapamycin. We also investigate Pim1's role as a target of aberrant somatic hypermutation (ASHM), the process by which the enzyme activation induced deamidase (AID) introduces mutations into the coding and non-coding regions of the Pim1 locus and other proto-oncogenes in many B-cell malignancies. We find that structural Pim1 mutants found in patient tumors samples retain their ability to accelerate tumorigenesis in the Eu-Myc model in vivo and to mediate resistance to apoptosis and rapamycin in vitro. In conclusion, Pim activity is clinically significant, highly oncogenic, and provides resistance to mTor inhibition. Pim inhibition is therefore an attractive therapeutic approach, especially in combination with PI3K/Akt/mTor inhibition, even as ASHM provides a potential mechanism for B-cell tumors to escape it.

Disclosures:

No relevant conflicts of interest to declare.
Clinical Significance of Lupus Anticoagulant and Anti-Beta2 Glycoprotein I Antibodies in a Pediatric Cohort.
Authors: Cook Rebecca P. , Marques Marisa , Oster Robert
Published: October 2019

Abstract 3989

Poster Board III-925

Introduction

Antiphospholipid antibodies (APAs) (lupus anticoagulant (LA), anti-beta2 glycoprotein I (anti-B2GPI) and anti-cardiolipin) are seen in a variety of conditions, yet their clinical implication varies. In adults, some APAs cause thrombosis and/or recurrent pregnancy loss. LAs with specificity for anti-B2GPI appear to be more closely associated with thrombosis. In children, APAs are often transient after viral or bacterial illnesses and little is known about their pathologic potential. In this study, we sought to determine the clinical associations of positive LAs with and without anti-B2GPI activity. Patients and Methods: We tested remnant plasma from 115 pediatric patients with positive LA result (according to the International Society on Thrombosis and Haemostasis guidelines) for anti-B2GPI IgG, IgM, and IgA using an indirect EIA supplied by REAADS (Corgenix Inc, Broomfield, CO) and reviewed the patient's medical records to correlate with the results.

Results

There were 61 females (53%) and 54 males (47%) with the average age of 10.6 years (range: 0.5 to 19.5 years). An underlying medical condition such as malignancy, autoimmune disorder, infection/sepsis, or concurrent thrombosis was observed in 97 patients (84%). Eighteen patients (16%) were healthy children and LA was detected incidentally during workup for a prolonged PTT. The mean age of the group with positive medical history (11.5 years; range: 0.5 to 13 years) was statistically different than that of the healthy children group which was 5.8 years (range: 0.5 to 3.75 years) (P<0.001). Of the 115 patients, 37 (32%) had thrombosis at the time of LA testing; only 6 patients had known inherited hypercoagulability. Thirty-four of the 37 thrombotic episodes (92%) were venous and 3 were arterial (8%). An autoimmune disorder was present in 46 patients (40%) and 10 developed a thrombosis. Forty-one patients (36%) had active infection/sepsis, and 15 had thrombosis. Of 12 patients with malignancies (10%), 4 had thrombosis. The average age of those with thrombosis, 13.3 years (range: 0.5 to 15 years) was higher than that of children without thrombosis, 10.3 years (range: 0.6 to 10.5 years) (P=0.018). Only 12 of 115 patients (10%) were positive for anti-B2GPI and 3 of them had venous thrombosis. Of the anti-B2GPI positive patients, 8 had underlying autoimmune conditions, 2 had no medical history but had current thrombosis, one had a malignancy with thrombosis, and another had an acute infection. Conclusions: In children, APAs occur in a variety of conditions, but may also be found incidentally. Perhaps surprising, LA was associated with thrombosis in 32% of patients. However, only 10% tested positive for anti-B2GPI, with an even smaller percentage having thrombosis (3%). These results suggest that anti-B2GPI is not necessary for the development of thrombosis in children with LA.

Disclosures:

No relevant conflicts of interest to declare.