bluuebunny/crossref_metadata_2025 huggingface dataset:
A Hyperactive Signalosome Results in High Sensitivity to HSP90 Inhibitors in AML
Authors: Zong Hongliang , Taldone Tony , Roboz Gail J. , Chiosis Gabriela , Guzman Monica L.
Published: October 2019

HSP90 is well established in supporting tumorigenesis by stabilizing oncogenic client proteins. Given this crucial role, a number of HSP90 inhibitors have been tested in various types of cancer, including leukemia. However, clinical trials thus far revealed only a subset of AML patients benefited from the treatment. Therefore, precision medicine approaches to define parameters that predict the patients' response to HSP90 inhibitors are needed to select patients who are most likely to benefit.

We have previously demonstrated that PU-H71, a novel purine scaffold HSP90 inhibitor with selectivity for a tumor-specific HSP90 and currently translating into Phase 2 clinical evaluation, is capable of ablating malignant blasts, progenitor and stem cells in AML patient samples using in vitro studies. We found that leukemia cell lines (n=18) and primary AML patient samples (n=26) with greater numbers of simultaneously activated signaling networks, including PI3K-AKT and JAK-STAT, were the most sensitive to HSP90 inhibition. Using different genetic models, our studies revealed that diverse oncogenic transformations that converge upon simultaneous hyperactivation of PI3K-AKT and JAK-STAT promote sensitivity to PU-H71.

To validate the efficacy of PU-H71 in vivo, we generated AML-GFP-luciferase xenograft models using cell lines with hyperactive signalosome. Xenotransplanted mice were treated with PU-H71 one week post-engraftment. In vivo imaging indicated that MOLM-13 xenografted leukemia was rapidly and significantly reduced by PU-H71 treatment. Six doses of PU-H71 produced robust anti-leukemic activity as indicated by in vivo imaging and flow cytometric analysis of post-treatment bone marrow (no disease detected). In addition, we generated 7 AML patient-derived xenografts (PDX) cohorts with samples that displayed varied levels of activation of PI3K-AKT and JAK-STAT signaling pathways. After initial validation that status of the PI3K-AKT and JAK-STAT signaling pathways were preserved in the PDX, we initiated treatment with PU-H71 and found that, as predicted, the AML-PDX with the most hyperactive signalosome were the most sensitive to in vivo treatment to PU-H71. Importantly, samples with hyperactive PI3K-AKT and JAK-STAT signaling also demonstrated a significant reduction in LSC using secondary transplants.

Taken together, we found that a hyperactive signalosome results in increased sensitivity to the HSP90 inhibitor PU-H71 in vitro and in vivo. Our study suggests that evaluation of PI3K-AKT and JAK-STAT signaling pathways may provide a means to select patients who are most likely to benefit from HSP90 inhibitory therapy.

Disclosures

No relevant conflicts of interest to declare.
Aberrant Expression of IL-22 Receptor 1 and IL-22 Stimulation Increase the Activation of STAT3 and ERK1/2 in ABC-DLBCL Cells
Authors: Lu Ting , Li Peng , Bi Chengfeng , Yu Shuang , Ma Daoxin , Fu Kai , Ji Chunyan
Published: October 2019

BACKGROUND: Interleukin-22 (IL-22) was recently discovered as a T-cell derived cytokine that belongs to the IL-10 gene family. It has been shown that IL-22 can promote cell proliferation and activate several oncogenic pathways including STAT3, MAPK and AKT signaling. IL-22 exerts its function by binding to its heterodimeric receptor complex which is composed of IL-22 receptor (R) 1 and IL-10R2. IL-22R1 is restricted and barely detected in normal immune cells including B and T cells. In the current study, we sought to determine whether IL-22 plays a part in the development of diffuse large B-cell lymphoma (DLBCL).

METHODS: Plasma IL-22 level was measured in newly diagnosed patients with DLBCL and normal individuals by ELISA assay. We also assessed the expression of IL-22R1 in FFPE sample and DLBCL cell lines (TMD8, OCI-LY3, SUDHL16, OCI-LY1) by immunohistochemistry and immunofluorescence staining respectively. Immunoblotting was performed to determine changes of signaling pathways in cell lines upon the treatment with IL-22 recombinant protein.

RESULTS: The plasma IL-22 concentration was significantly increased in newly diagnosed DLBCL patients (n=12) compared with normal individuals (n=26, P= 0.0177). IL-22R1 protein was expressed in all of the four DLBCL cell lines, while not all but some of the primary tumor samples expressed it on membranes of tumor cells. Using immunoblotting, we demonstrated a substantial increase of pSTAT3 level with the treatment of IL-22 in ABC-DLBCL cell lines TMD8 and OCI-LY3, while no pSTAT3 was detectable in GCB-DLBCL cell lines SUDHL16 and OCI-LY1. Interestingly, upon IL-22 stimulation, the p-ERK1/2 level was increased shortly and quickly returned to the normal level in TMD8 cells, and steadily increased in LY3 cells during 30 min of treatment. However, there was no change about p-ERK1/2 in SUDHL16 and OCI-LY1 cells after IL-22 treatment.

CONCLUSIONS: Our current study has shown the aberrant expression of IL-22R1 in DLBCL. And recombinant IL-22 protein increased the activation of STAT3 and ERK1/2 in ABC rather than GCB-DLBCL cell lines. For the first time, we suggested the importance of IL-22 and its receptor IL-22R1 pathway in ABC-DLBCL, which warrants further studies.

Figure 1. The expression of IL-22R1 in DLBCL cell lines was detected by using immunofluorescence staining.

Disclosures

No relevant conflicts of interest to declare.
Gender and Obesity Do Not Impact Survival in Diffuse Large B-Cell Lymphoma: Justification for Full Weight-Based Chemotherapy Dosing in All Patients
Authors: Nikonova Anna
Published: October 2019

Background: The addition of rituximab to CHOP chemotherapy has greatly improved outcomes in diffuse large B-cell lymphoma (DLBCL). However, subgroup analyses from prospective trials have introduced the question of differential benefit of rituximab in older females, due to slower drug clearance (Müller C1 et al. Blood. 2012; 119(14):3276-84; Zeynalova S, et al. Blood. 2014; 123(5):640-6.) A related question has been the interplay of gender and obesity on outcomes in DLBCL.

Methods: We evaluated the impact of gender and obesity on overall and progression-free survival and treatment toxicity in DLBCL. We further studied the impact of weight loss over the chemotherapy course on outcomes. A comprehensive clinical database of 490 patients with a new diagnosis of de novo or transformed DLBCL treated at our center between 2002 and 2013 was used. All patients received at least one cycle of R-CHOP chemotherapy (median 6). We defined older age as greater then 60 and obesity as BMI >25. Chemotherapy toxicity was defined as any admission for febrile neutropenia or other serious infection.

Results: In the population studied (n=490), the median age was 64, with 60% over age 60, and 49.4% were female. There were 51% of patients with BMI >25. All patients received full weight-based chemotherapy dosing, except for universal vincristine dose capping at 2 mg. A total of 173 admissions for febrile neutropenia or other serious infection were identified. Median overall survival and progression-free survival follow up times were 39 and 38 months respectively. The two groups: obese and non-obese, were well balanced with respect to age, gender, ECOG, B-symptoms, and R-IPI. On Cox multivariable regression analysis, only R-IPI (HR=16; p<0.001), Charlson comorbidity index (HR=4.7; p=0.029) and presence of B-symptoms (HR=6.5; p=0.011) impacted overall survival. In the analysis for progression-free survival, only R-IPI (HR 7.7, p=0.005) and weight loss of > 10% over the full treatment course (HR =4.6; p=0.33) were prognostic. In the logistic regression analysis of chemotherapy toxicity, only ECOG performance status was predictive for infectious complications (OR=6; p=0.14).

Conclusions: In this large retrospective cohort of patients with DLBCL, we could not find an association between gender - in either younger or older subgroups - and lymphoma survival. Weight loss during treatment represented an important prognostic marker of long-term outcome. However, patients who were obese at baseline and received full weight-based chemotherapy dosing did not experience increased toxicity or worse long-term outcomes. These represent the first available data in lymphoma to support recent broad recommendations to forgo chemotherapy dose capping in obese patients.

Figure 1. Figure 1. Figure 2. Figure 2. Figure 3. Figure 3.

Disclosures

No relevant conflicts of interest to declare.
Biomarker Predictive Ibrutinib Response Using Profiled ABC-DLBCL Patient Derived Xenografts
Authors: Wu Ran , Guo Sheng , Cai Jie , Huang Xuesong , Yang Jie , Wery Jean-Pierre , Li Henry Qi-xiang
Published: October 2019

Background. Activated B cell-like (ABC) is a subtype of DLBCL difficult to treat. Activation of B-cell receptor (BCR) signaling pathway, including CD79 transmembrane proteins (A/B subunits) and Bruton's tyrosine kinase (BTK), are required for the disease maintenance. The MYD88 (an adapter for Toll-like receptors in a separate pathway from BCR pathway) activating mutation, L265P, has been suggested to activate BCR pathway (29% of ABC-DLBCL) and thus drives the disease. These DLBCLs seem responsive to ibrutinib (a BTK inhibitor) in the clinics1,2. Majority of MYD88-L265P mutant DLBCL patients also have CD79B mutation (e.g. Y197N), i.e. double mutants (21% of total ABC-DLBCL), hinting collaborative oncogenesis, while some are MYD88-L265P single mutant with wild type CD79B. It remains unclear whether the single and double mutant ABC-DLBCL differ in their molecular pathogenesis as well as response to ibrutinib.

Method. We created DLBCL patient derived xenografts (PDX) as experimental system mimicking patients in order to investigate the molecular pathogenesis/response to ibrutinib. We first transcriptome-sequenced the established PDXs. We categorized them into ABC/GCB subtypes along with mutation status of CD79B/MYD88 per transcriptome profiles. We then assessed these PDXs for their response to ibrutinib and trying to correlate antitumor activity to molecular signature.

Results. We have created 20 NHL-PDX, with 5 of them transcriptome-sequenced. Clustering analysis per expression profile of 14-genes have classified all these 5 into subclass ABC-DLBCL. Among the 5, one is MYD88-L265P/CD79B-Y197N double mutants (LY2298), one MYD88-L265P single mutant (LY0257), and three wild types (LY2345, LY2214, LY2266). Among the 3 models tested so far, LY2214 (wildtype) and LY0257 (single mutation) did not respond to ibrutinib, but LY2298 (double mutations) did. These results, although small sample sizes, seem to suggest that double mutations are required for the response to ibrutinib, while single mutant and wild type does not. These observation seem to be consistent with the implication from recent clinical study1. We are currently profile and test the remaining models to further confirm these observations. We will also further elucidate the downstream signaling along with the drug response.

Conclusion. Our preliminary results confirmed that ibrutinib sensitivity correlates with the concomitant double mutation of CD79B and MYD88.

References

1. Wilson, W.H., et al. Targeting B cell receptor signaling with ibrutinib in diffuse large B cell lymphoma. Nature medicine (2015).

2. Aalipour, A. & Advani, R.H. Bruton's tyrosine kinase inhibitors and their clinical potential in the treatment of B-cell malignancies: focus on ibrutinib. Therapeutic advances in hematology5, 121-133 (2014).

Disclosures

No relevant conflicts of interest to declare.
Activation of Notch-Hes Signaling Pathway Impairs Osteogenic Differentiation and Hematopoietic Stem Cell-Supporting Capacitiy of Mesenchymal Stromal Cells Derived from Myelodysplastic Syndromes Patients
Authors: Chang Chunkang , Fei Chengming , Guo Juan , Zhao Youshan , Gu Shucheng , Li Xiao
Published: October 2019

Ineffective hematopoiesis is a major characteristic of myelodysplastic syndromes (MDS). Bone marrow mesenchymal stromal cells(BMMSCs) and their progeny (i.e., osteoblasts, adipocytes, and reticular cells), which are considered as main cellular components of the bone marrow niche, have been shown to physiologically support hematopoiesis, but their contribution to the pathogenesis of MDS is controversially discussed.

In this study, we examined the osteogenic differentiation and hematopoietic stem cell-supporting capacitiy of BMMSCs in patients with MDS (n=67) and healthy donors (n=22). After 21 days osteogenic induction differentiation, osteogenesis potential of BMMSCs was significantly reduced in cases with RARS(83.3%), RCMD(75.0%), RAEB I(44.4%), RAEB II (40%), indicated by cytochemical stainings and reduced expressions of Runx2. Moreover, we observed that in co-cultures with normal hematopoietic stem cells(HSCs) and MDS-BMMSCs, the colony number (CFU-GM、BFU-E and CFU-GEMM) was significantly lower in the presence of MDS-BMMSCs in comparison to the normal counterpart.

Furthermore, in MDS-BMMSCs, we detected increased mRNA expression of several members of the Notch pathway, including Delta-like-1, Jagged-1, Notch1, Notch2, Hes1 and Hes5. Basically, the Notch-Hes pathway is the main regulator of the microenvironment dependent hematopoietic stem cell fate. Therefore we investigated if the activation of Notch-Hes pathway affected their osteogenesis and hematopoietic stem cell-supporting capacitiy of BMMSCs. By overexpression of Notch1 intracellular domain (NICD) in BMMSCs from healthy donors, we confirmed that Notch signaling negatively regulated BMMSCs osteogenesis through inhibition of Runx2 transcriptional activity. Importantly, treatment with the Notch1 inhibitor DAPT reversed the osteogenic differentiation and improved the hematopoiesis supporting capacitiy of MDS-BMMSCs.

Taken together, our findings suggest that the ineffective hematopoiesis typical of MDS may be partly due to the impaired osteogenic differentiation of BMMSCs, and the activation of Notch-Hes signaling is involved in the impaired osteogenic differentiation and diminished hematopoietic stem cell-supporting capacitiy of MDS-BMMSCs, restoring the adequate Notch-Hes signaling could represent a potential therapeutic approach to MDS.

Disclosures

No relevant conflicts of interest to declare.
Immunosuppressive FVIII-Specific Human Cartregs in Hemophilia a
Authors: Yoon Jeong Heon , Schmidt Anja , Kim Yong Chan , Koenigs Christoph , Scott David William
Published: October 2019

Hemophilia A is an X-linked disorder, in which mutations in the coagulation Factor VIII (FVIII) gene lead to a loss of FVIII function and serious bleeding episodes. These episodes can be treated with recombinant FVIII protein replacement. Unfortunately, ~25% of hemophilia A patients produce inhibitory anti-FVIII antibodies because of lack of tolerance. Thus, it is necessary to develop effective tolerogenic therapies to prevent, as well as reverse, inhibitor formation. Previously, we generated engineered antigen-specific regulatory T cells (Tregs), created by transduction of a recombinant T-cell receptor (TCR) isolated from a hemophilia A subject's T cell clone. The resulting engineered T cells bind MHC tetramers, proliferate in response to a specific FVIII epitope, and suppress effector responses to FVIII.

In this study, we engineered a FVIII-specific chimeric antigen receptor (ANS8CAR) using a FVIII-specific scFv derived from a synthetic phage display library. Following initial experiments in naïve CD4 T cells, this CAR was introduced into human Tregs. Western blot and specific staining with FVIII verified CAR expression. Transduced ANS8CAR Tregs proliferated in response to FVIII and were able to suppress the proliferation of FVIII-specific T effector cells in vitro. Additionally, the proliferation of T effector cells with different FVIII domain specificity was suppressed as well when ANS8CAR-transduced Tregs were activated with FVIII. Thus, engineered cells are able to promote bystander suppression. Cytokine expression of ANS8CAR-transduced Tregs was comparable to expression of untransduced and TCR-transduced Tregs indicating that the regulatory phenotype of Tregs was not negatively influenced by ANS8CAR expression.

In conclusion, CAR-transduced Tregs seem to be a promising alternative to TCR-transduced Tregs for a future tolerogenic treatment of hemophilia A patients with inhibitory FVIII-specific antibodies.

Supported by NIH grants HL061883 and HL126727 (DWS), the Society of Thrombosis and Hemostasis Research, and the Günter Landbeck Excellence Award (AS).

Disclosures

Kim: Henry Jackson Foundation: Other: patent filed. Scott:Henry Jackson Foundation: Other: patent filed.
Identification of B Cell Receptor Antigens in the Chronic Lymphocytic Leukemia Microenvironment
Authors: ten Hacken Elisa , Oellerich Thomas , Gounari Maria , Hoellenriegel Julia , Pan Kuan-Ting , O'Brien Susan , Wierda William , Ferrajoli Alessandra , Estrov Zeev , Keating Michael J. , Urlaub Henning , Ghia Paolo , Burger Jan A.
Published: October 2019

Background: B cell receptor (BCR) signaling is a central pathway in Chronic Lymphocytic Leukemia (CLL) pathogenesis that is activated by interactions between CLL cells and the microenvironment in secondary lymphoid organs. Nurselike cells (NLCs) are an important component of this microenvironment, and co-culture of CLL cells with NLCs activates BCR signaling. CLL BCRs are able to recognize vimentin and calreticulin proteins exposed on the surface of NLCs and these interactions are responsible for stromal-mediated anti-apoptotic effects. However, the exact mechanism of BCR activation and the nature of the BCR ligands expressed by NLCs still remain incompletely defined.

Aim: The aim of this project is to identify and validate ligands expressed by NLCs that activate BCRs on CLL cells.

Methods: CLL PBMCs from 3 CLL patients were cultured in vitro for 14 days until outgrowth of NLCs. Then, NLCs were harvested and lysed, followed by immunoprecipitation with recombinant monoclonal antibodies obtained from 4 different CLL patients carrying unmutated IGHV genes (U-CLL). Immunoprecipitation of human hTERT mesenchymal stromal cells was used as a negative control. Immunoprecipitated proteins were analyzed by label-free quantitative mass spectrometry followed by bioinformatic data analysis using the softwares MaxQuant and Perseus. The quantitative mass spectrometric data enabled us to distinguish between unspecific background proteins and putative BCR ligands.

Results: In all samples, around 2600 proteins were identified and around 2000 of them were quantified using mass spectrometry. Unsupervised hierarchical clustering identified the enrichment patterns of NLC-derived BCR ligands. We identified 6 different protein clusters; among them, one cluster included 11 putative CLL BCR antigens with a fold-change cut-off above 10, which were enriched in all 3 NLC samples, but not in hTERT cells. These BCR ligands included cytoskeletal proteins, ER-associated proteins, and membrane-associated proteins, some of them with known auto-antigenic function in other diseases.

Conclusion: Recombinant BCRs from U-CLL patients recognize a large number of proteins expressed by NLCs, identified through immunoprecipitation of NLC lysates with CLL BCRs, followed by label-free mass spectrometry. The identified ligands will be further validated by epitope-mapping and BCR activation functional studies to allow a better characterization of the pathogenic antigens in CLL, and of the mechanisms driving CLL survival in the tissue microenvironment.

Disclosures

Wierda: Glaxo-Smith-Kline Inc.: Research Funding; Celgene Corp.: Consultancy. Estrov:incyte: Consultancy, Research Funding. Burger:Pharmacyclics LLC, an AbbVie Company: Research Funding.
BAR-CD8 T-Cell Mediated Targeted Killing of Inhibitor Producing FVIII-Specific B Cells
Authors: Parvathaneni Kalpana , Zhang Aihong , Kim Yong Chan , Scott David William
Published: October 2019

Anti-FVIII antibody (inhibitor) formation is a major complication in the treatment of Hemophilia A. Our lab has focused on developing novel approaches to modulate inhibitor formation by regulation of FVIII-specific T effectors that recognize FVIII and "help" B cells to produce antibodies to FVIII. As an alternative approach, in this presentation, we directly target anti-FVIII B cells. Therefore, we have developed a novel chimeric antigen-like receptor called B-cell antibody recognizing receptor (BAR) to target and directly kill the FVIII-specific B cells and antiobody-producing cells . Our BAR comprises of FVIII-domain C2 attached to T cell signaling CD28 and CD3ζ elements. We reasoned that CD8 T cells transduced to express the FVIII C2 should trigger cytotoxicity upon recognition by and interaction with specific B cells. We expressed the C2-BAR or Ova-BAR (control) in human T cells and demonstrated expression by flow cytometry. These BAR CD8 T cells also proliferated upon addition of anti-C2 or anti-Ova, respectively post 72 hrs. For proof of principle that these BAR-expressing CD8 T cells can kill specific B cells, the following basic experiment was designed. Mouse CD8 T cells transduced to express C2-BAR were co-cultured with FVIII-specific hybridomas (expressing surface IgG anti-C2) at a various ratios. After 5 hours, we measured antibody secretion by ELIspot assay. Our results demonstrated suppression of antibody secretion by C2 BAR expressing CD8 T cells, but not by control Ova-expressing BAR CD8s. These results provide evidence that BAR CD8 T cells expressing FVIII domains can be used to target specific B cells to suppress inhibitor formation, and may also provide a platform to treat other undesirable antibody responses. (Supported in part by a Pfizer ASPIRE award and NIH grant HL061883)

Disclosures

Zhang: Henry Jacksopn Foundation: Other: patent filed. Kim:Henry Jackson Foundation: Other: patent filed. Scott:Henry Jackson Foundation: Other: patent filed.
UCHL1 Is a Biomarker of Aggressive Multiple Myeloma Required for Disease Progression
Authors: Hussain Sajjad , Bedekovics Tibor , Chesi Marta , Bergsagel Leif , Galardy Paul J.
Published: October 2019

The success of proteasome inhibition in multiple myeloma highlights the critical role for the ubiquitin-proteasome system (UPS) in this disease. However, there has been little progress in finding more specific targets within the UPS involved in myeloma pathogenesis. In an unbiased screen of de-ubiquitinase activity, we previously found the ubiquitin hydrolase UCH-L1 to be frequently over-expressed in B-cell malignancies, including myeloma. Using a transgenic mouse model, we also found that aberrant expression of UCH-L1 is sufficient to drive the development of spontaneous B-cell lymphomas and plasmacytomas, demonstrating its oncogenic activity. Whether and how UCH-L1 affects the clinical behavior of myeloma, however, is not known. Here we show that UCH-L1 is a potent negative prognostic factor that is essential for the dissemination and progression of myeloma. We found high levels of UCHL1 to predict early progression in newly diagnosed patients; a finding reversed in studies including bortezomib. We also found high UCHL1 levels to be a critical factor in the superiority of bortezomib over high-dose dexamethasone in relapsed patients. High UCHL1 partially overlaps with, but is distinct from, known genetic risks including 4p16 rearrangement and 1q21 amplification. Using an orthotopic mouse model, we found UCH-L1 depletion delays myeloma dissemination and causes regression of established disease. We conclude that UCH-L1 is a biomarker of aggressive myeloma that may be an important marker of bortezomib response, and may itself be an effective target in disseminated disease.

Disclosures

Galardy: Mission Therapeutics: Research Funding.
Pharmacogenomics of Bortezomib in Multiple Myeloma Patients Reveals That the Ubiquitin Ligase SCF-Skp2 Promotes Drug Resistance
Authors: Driscoll James J. , Malek Ehsan
Published: October 2019

The ability to couple cancer patients with genetically-targeted therapeutic strategies remains a major challenge due to the heterogeneous and evolvable nature of individual tumors. This is particularly true of the molecularly diverse and complex disease multiple myeloma (MM). Clinical efficacy of the proteasome inhibitor (PI) Bortezomib (VelcadeTM) established the Ub+proteasome system (UPS) as a key target in MM. While the therapeutic benefit of PIs in MM remains unchallenged, many patients do not respond to therapy and drug resistance develops nearly uniformly. Despite marked therapeutic advances for most patients with MM, outcomes remain poor for 15% to 20% of patients presenting with high-risk disease defined by host-related and tumor-related factors and expert guidelines recommend enrollment in a clinical trial for the majority of patients who will ultimately fail approved therapies.The Skp1-Cul1-F-box protein (SCF) ubiquitin ligase SCFSkp2 promotes ubiquitination and degradation of the cell-cycle inhibitor p27. Increased SKP2 expression and reduced p27 levels are frequent in human cancers and are associated with therapeutic resistance and poor prognosis. SCFSkp2 ubiquitination activity is increased by Commd1, a copper metabolism MURR1 domain-containing 1 protein that binds Cul1 and by Cks1B that increases binding of phosphorylated p27 to Skp2. Bortezomib is a proteasome inhibitor that has remarkably improved multiple myeloma (MM) patient outcome, but dose-limiting toxicities and development of resistance limit its long-term utility. Here, we observed higher CUL1 and COMMD1 mRNA levels in Bortezomib-resistant patient MM cells and correlated higher CUL1, COMMD1, SKP2 and CKS1B levels with decreased survival. Genetic knockdown of CUL1 or COMMD1 disrupted the SCFSkp2 complex, stabilized p27, synergistically increased the generation of annexin-positive cells by bortezomib and enhanced the anti-myeloma effect of PIs against resistant cells. Chemical library screening identified a novel compound, DT204, that reduced Skp2 association with Cul1 and Commd1, inhibited p27 degradation and induced synergistic anti-MM activity combined with Bortezomib in patient tumor cells and chemoresistant stem cell-like cells to overcame Bortezomib resistance. Bortezomib treatment with DT204 also reduced the growth of xenotransplanted myeloma cells in mouse models and prolongs host survival. Taken together, our results demonstrate that the SCFSkp2 -p27 axis represents a major determinant in the ability of Bortezomib to induce apoptosis in MM and provide proof-of-concept for the combination of SCFSkp2 inhibitors with Bortezomib as a strategy to overcome Bortezomib resistance.

Disclosures

No relevant conflicts of interest to declare.
Prenatal Allospecific Tolerance Is Characterized By Early Treg Expansion and a Surprisingly Slow Pace for Teff Clonal Deletion
Authors: Strong Beverly SI , Lee Amanda E , Newkold Tess J , Turner Lucas E , Shaaban Aimen F
Published: October 2019

Prenatal transplantation capitalizes on the unique fetal environment, allowing for life-long engraftment of allogeneic stem cells without the need for harsh conditioning regimens. A prerequisite for stable engraftment of allogeneic cells likely requires the negative selection of donor-specific host effector T cells (Teff) and the support of donor-specific regulatory T cells (Tregs). However, little is known about the interplay between these cell types during development. The purpose of this study was to characterize the dynamic relationship between donor-specific Teff and Tregs as they emerge during development.

Prenatal allogeneic chimeras were established by in utero transplantation of E14 fetal liver light density cells into age-matched allogeneic fetal recipients (Balb/c to B6 or B6 to Balb/c). In this model, immature T cells from B6 mice expressing TCRv-beta-5, 11, and 12 are negatively selected by mtv-8 superantigen complexed with I-E class MHC II on Balb/c cells. As alpha/beta TCR rearrangement does not occur until E16, this established transplantation model allows for alloantigen to be present from the earliest stages of thymic selection. Kinetic analysis of donor-specific T cell populations was performed in peripheral blood paired with in depth analysis in thymus and spleen in control and chimeric mice.

Negative selection of donor-specific Teff cells occurs at an unexpectedly slow pace in Balb/c to B6 prenatal chimeras. Donor-specific CD4 and CD8 Teff are significantly decreased in frequency at 4 weeks of age but do not reach maximal deletion until 12 weeks of age (TCRv-beta-5 data shown in Figure A). Further analysis demonstrated that this slow elimination of donor-specific Teff was paired with an early increase in the frequency of donor-specific Tregs at 4 weeks of age (TCRv-beta-5 data shown in Figure B.) This increase in donor-specific Tregs likely occurred as a result of peripheral expansion as there was no change in the frequency of donor-specific Tregs in the thymus (Figure B) and no change in the frequency of these cells that expressed the markers of thymically derived natural Tregs neuropilin-1 or helios (data not shown). In agreement with this hypothesis, donor-specific splenic Tregs incorporated BrdU at a higher rate than other Tregs in young mice indicating a potential expansion of donor-specific Tregs in the periphery (TCRv-beta-5 data shown in Figure C.)

Collectively, these data demonstrate that prenatal transplantation is characterized by: 1) a surprisingly slow reduction of donor-specific Teff subsets; 2) an early expansion of donor-specific Tregs. Further studies will explore the role of donor-specific Tregs in controlling early immunity to prenatally encountered antigens.

Figure 1. Figure 1.

Disclosures

No relevant conflicts of interest to declare.
RIG-I-Induced Type I IFNs Promote Regeneration of the Intestinal Stem Cell Compartment during Acute Tissue Damage
Authors: Fischer Julius C , Bscheider Michael , Eisenkolb Gabriel , Wintges Alexander , Lindemans Caroline A , Heidegger Simon , Monette Sebastien , Calafiore Marco , Porosnicu Rodriguez Kori A. , Lieberman Sophie , Liu Chen , Peschel Christian , Docampo Melissa , Velardi Enrico , Jenq Robert R. , Hanash Alan M , Dudakov Jarrod A , Haas Tobias , van den Brink Marcel , Poeck Hendrik
Published: October 2019

Introduction: Although the role of Type I IFNs in initiating host defense against pathogens is well established, recent work highlights the regenerative function of this cytokine family. Yet, despite its involvement in tissue repair, the cellular targets and mechanisms of action by which Type I IFNs act during tissue regeneration are poorly understood.

Results: Here, we describe a hitherto unrecognized regenerative function for the RIG-I/MAVS/IFN-I pathway through direct effects on epithelial regeneration. Mice deficient in the RIG-I adaptor MAVS were more sensitive to intestinal barrier damage after total body irradiation (TBI) and, like RIG-I deficient mice, developed worse graft-versus-host disease (GVHD) in a preclinical model for allogeneic hematopoietic stem cell transplantation (allo-HSCT). This phenotype was not associated with changes in the intestinal microbiota, but with a defect in epithelial regeneration. Moreover, in contrast to previous reports in steady-state conditions and after viral challenge, we found that interferon-α/β receptor (IFNAR) signaling in non-hematopoietic epithelial cells was crucial for tissue regeneration after acute damage. Importantly, we could demonstrate that this pathway could be therapeutically targeted with RIG-I agonists, which promoted barrier integrity and prevented GVHD. Mechanistically, Type I IFNs (either RIG-I-induced or recombinant) could promote intestinal stem cell (ISC) growth in crypt organoid cultures, suggesting that stimulation of the ISC compartment led to epithelial regeneration.

Conclusion: Our findings suggest that activation of RIG-I and IFN-I can promote regeneration of intestinal epithelial cells and thus offers an innovative therapeutic strategy for the management of acute intestinal injury.

Disclosures

van den Brink: Merck: Honoraria; Boehringer Ingelheim: Consultancy, Other: Advisory board attendee; Regeneron: Honoraria; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tobira Therapeutics: Other: Advisory board attendee.
Outcomes of Grades II-IV Acute Graft-Versus-Host Disease Post-Allogeneic Hematopoietic Stem Cell Transplantation: How Much Progress Was Achieved?
Authors: Khoury Hanna Jean , Wang Tao , Arora Mukta , Hemmer Michael , Spellman Stephen R. , Cutler Corey S , Couriel Daniel R. , Alousi Amin M. , Joseph Joseph A.
Published: October 2019

Despite recent reduction in mortality following hematopoietic cell transplant (HCT), graft versus host disease (GVHD) remains a major causes of failure. It is unclear if advances in antiviral, antifungal and antibacterial prophylaxis/treatment, and immunosuppression, have led to better survivals following the onset of acute GVHD (aGVHD). We therefore analyzed within the CIBMTR database outcomes over time of allogeneic HCT recipients who developed grade II-IV aGVHD. The objectives were to determine the change in overall survival (OS), disease-free survival (DFS), treatment-related mortality (TRM) and to identify risk factors.

Methods: Between 1999 and 2012, 7295 patients with AML (57%), ALL (29%) or MDS (14%) received sibling (35%) or unrelated donor (65%) blood (65%) or marrow (35%) HCT (1216 for 1999-2001, 2329 for 2002-2005, 3750 for 2006-2010), and 3312 (45%) developed grades II-IV aGVHD. Median follow-up was 141 (3-174), 96 (3-144) and 49 (3-95) months for 1999-2001, 2002-2005, and 2006-2010 periods respectively. Multivariate models were built using the Cox's proportional hazards model. Clinical variables were tested for the affirmation of the proportional hazards assumption. Factors violating the proportional hazards assumption were adjusted through stratification. Stepwise model building procedure was used in developing models for each outcome with a threshold of 0.05 for both entry and stay in the model. Interactions between the main variable, (i.e. year of transplant) and the adjusted covariates were tested.

Results: Univariate analyses showed a decrease in the incidence of grades III-IV aGVHD: 56 % (52-60), 47 %(44-50), and 38% (36-40) for 1999-2001, 2002-2005, and 2006-2010, respectively (p= <0.001). Multivariate analyses showed that age of recipient, organ affected by aGVHD, treatment of aGVHD (steroids vs. other), disease status at transplant and KPS influenced OS; while age of recipient, organ affected by aGVHD, treatment of aGVHD (steroids vs. other), CMV serostatus matching, disease status at transplant, unrelated donor age and KPS affected OS and TRM. After adjusting for these factors, recipients of tacrolimus-based GVHD prophylaxis who developed grade II aGVHD had better OS after 2006 (HR 0.78, p=0.02), while a trend for lower TRM (HR 0.79, p=0.056) and better OS (HR 0.83, p=0.08) were observed for those with grades III-IV aGVHD.

Conclusions: Incidence of grades III-IV aGVHD has overall decreased and OS and TRM have improved over time for recipients of tacrolimus-based GVHD prophylaxis who developed grades II-IV aGVHD.

Disclosures

Alousi: Therakos, Inc: Research Funding.
The Long Non-Coding RNA NEAT1 Modulates Imatinib-Induced Apoptosis in CML Cells
Authors: Zeng Chengwu , Liu Sichu , Yang Lijian , Chen Shaohua , Li Yangqiu
Published: October 2019

Chronic myeloid leukemia (CML) is a clonal disease characterizedby the presence of a constitutively active tyrosine kinase BCR-ABL oncoprotein. Although BCR-ABL is crucially important for pathogenesis and responsiveness to treatment, it is thought that some additional factors might involve in regulation in this process. For example, aberrant expression of Long noncoding RNAs (lncRNAs) have recently been identified to be involved in various diseases including cancer, suggesting that lncRNAs may play a role in BCR-ABL-mediated CML. In order to characterize the effect of lncRNAs in CML, we firstly performed lncRNA expression profiling microarray in newly diagnosed CML patients and healthy individuals¡¯ samples. We found that nuclear enriched abundant transcript1 (NEAT1), an lncRNA essential for the formation of nuclear body paraspeckles, is significantly repressed in in CML patients samplescompared with those of healthy donors (figure 1A). We found that NEAT1 expression was repressed by BCR-ABL, and the expression of NEAT1 could be restored after treatment with imatinib and BCR-ABL mediated pathway inhibitor or knockdown BCR-ABL in K562 cells (figure 1B). Moreover, knockdown of NEAT1 combining with imatinib treatment, could promote apoptosis of K562 cells (figure 1C). Importantly, the NEAT1-binding paraspeckle protein splicing factor proline/glutamine-rich (SFPQ) is required for NEAT1-mediated apoptosis of K562 cells. Furthermore, NEAT1 was shown to be regulated by c-Myc in K562 cells (figure 1D). Taken together, these results are the first to assign a biological function to the nuclear long noncoding RNANEAT1 inapoptosis CML cells and may lead to a fuller understanding of the molecular events leading to CML.

Figure 1. Figure 1.

Disclosures

Zeng: National Science Foundation of China (No. 81400102) and China Postdoctoral Science FoundationiNo. 2015M570751): Research Funding. Li:National Science Foundation of China (No. U1301226): Research Funding.
High BCR-ABL/GUSIS Levels at Diagnosis Are Associated with Unfavorable Responses to Standard Dose Imatinib
Authors: Vigneri Paolo , Stagno Fabio , Stella Stefania , Cupri Alessandra , Forte Stefano , Massimino Michele , Antolino Agostino , Caracciolo Clementina , Nocilli Laura , Impera Stefana , Musolino Caterina , Turri Diamante , Russo Mario , Tomaselli Carmela Anna Maria , Rizzo Michele , Musso Maurizio , Morabito Fortunato , Levato Luciano , Manzella Livia , Mueller Martin C , Hochhaus Andreas , Di Raimondo Francesco
Published: October 2019

Background

The approval of second-generation tyrosine kinase inhibitors (TKIs) for the first line treatment of Chronic Myeloid Leukemia (CML) has generated a need for early molecular parameters associated with inadequate responses to Imatinib Mesylate (IM).

Objective

We correlated quantitative determination of BCR-ABL transcripts at diagnosis with the outcome (defined according to the 2013 European Leukemia Net recommendations) of 272 newly diagnosed CML patients receiving IM 400 mg/die.

Methods

BCR-ABL transcripts were measured from peripheral blood samples drawn at diagnosis before patients received any pharmacological treatment using Real-Time Quantitative PCR (RQ-PCR). All molecular determinations were performed twice (in triplicates) on the same sample using either ABL or glucuronidase-beta (GUS) as reference genes. BCR-ABL values were then reported on the international scale (IS).

Results

With a median follow-up of 60 months, 65.4% of patients achieved an optimal response, 5.6% presented a response currently defined as "warning", 22.4% failed IM treatment and 6.6% switched to a different tyrosine kinase inhibitor because of intolerance to the drug. We recorded 19 deaths (6.9%), 7 (2.5%) attributable to disease progression. We applied Receiver Operating Characteristic (ROC) curves to define BCR-ABL/GUSIS expression levels that would separate patients likely (i.e. below the threshold) or unlikely (i.e. above the threshold) to achieve multiple endpoints, namely: optimal response (OR), failure-free survival (FFS), event-free survival (EFS), transformation-free survival (TFS) and overall survival (OS). Employing the specific threshold calculated for each endpoint we found that high BCR-ABL/GUSIS levels at diagnosis were associated with inferior probabilities of OR (p<0.001), FFS (p<0.001) and EFS (p<0.001). Elevated BCR-ABL/GUSIS levels were also associated with higher rates of disease transformation to the accelerated phase or blast crisis (p=0.029) but not with OS (p=0.132).

Conclusions

High BCR-ABL transcripts at diagnosis measured by RQ-PCR employing GUS as a reference gene allow the identification of CML patients unlikely to benefit from standard dose IM that should be considered for alternative forms of treatment.

Disclosures

Hochhaus: Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; ARIAD: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding.
HH-002 Effectively Suppresses Myeloproliferative Neoplasm Phenotypes in JAK2V617F Transgenic Mice
Authors: Ho Wan-Ting , Zhao Wanke , Hallenbeck Paul , Rong Frank , Zhao Zhizhuang Joe
Published: October 2019

Ph-negative myeloproliferative neoplasms (MPNs) represent a group of conditions characterized by chronic increases in some or all of the blood cells (platelets, white blood cells, and red blood cells). A major molecular defect in MPNs is JAK2V617F, a gain-of-function mutant form of tyrosine kinase JAK2 found in the majority of MPN patients. In earlier studies, we generated JAK2V617F transgenic mice by using the vav promoter which drives gene expression in all hematopoietic cells. These mice develop MPN-like phenotypes in a transgene dose-dependent manner. The objective of this study is to use this animal MPN model to test the efficacy of HH-002, a derivative of natural plant alkaloid homoharringtonine.

Treatment of JAK2V617F transgenic mice with a daily dose of 1 mg/kg HH-002 through subcutaneous injection reduced blood cell counts to the normal range or slightly below the normal level. HH-002 causes a preferential reduction of myeloid cells in JAK2V617F transgenic mice since percentages of lymphocytes (CD3e+ T cells and CD19+ B cells) were increased (despite somewhat decreases in absolute numbers) while the percentage of Gr-1+/CD11b+ granulocytes sharply declined. HH-002 also effectively reduced the spleen size of JAK2V617F transgenic mice and prevented development of myelofibrosis. With a JAK2V617F bone marrow transplant mouse model, HH-002 showed a similar effect. Although it did not increase the ratio of JAK2V617F-negative cells to JAK2V617F-positive, it stopped further expansion of JAK2V617F-containing malignant cells in JAK2V617F bone marrow recipient mice. In vitro experiments with primary hematopoietic cells demonstrated that HH-002 potently inhibited formation of erythroid and myeloid colonies with an IC50 value of 1-3 nM. However, it does not show a preferential inhibition of JAK2V617F-containing cells. This is not unexpected since homoharringtonine is known to inhibit protein synthesis. Taken together, HH-002 is a promising candidate for development of therapeutic drugs to treat MPNs and related diseases. Combination drug therapies with JAK2 inhibitors need to be explored.

Disclosures

Hallenbeck: Hangzhou Bensheng Pharmaceutical Corp. Ltd.: Employment, Equity Ownership. Rong:Hangzhou Bensheng Pharmaceutical Corp. Ltd.: Employment, Equity Ownership. Zhao:Hangzhou Bensheng Pharmaceutical Corp. Ltd.: Research Funding.
A Patient-Derived EZH2 Mutant Induces MDS-like Diseases with Derepressed ABCG2 Expression in Mice
Authors: Kawabata Kimihito Cojin , Inoue Daichi , Kitaura Jiro , Harada Yuka , Goyama Susumu , Harada Hironori , Aburatani Hiroyuki , Kitamura Toshio
Published: October 2019

A histone H3 Lysine 27 (H3K27)-methyltransferase, enhancer of zeste homolog 2(EZH2) is known as a tumor-associated gene. Physiological role of EZH2 is an enzymatic component of polycomb repressive complex 2 (PRC2) to inhibit expression of target genes. While EZH2 plays oncogenic roles by repressing the expression of tumor suppressors in solid tumors and some lymphomas, it plays rather tumor-suppressive roles in myeloid malignancies. We have generated a short-form EZH2 that lacks the catalytic SET domain (EZH2-dSET). Using this EZH2 mutant we could produce serially transplantable MDS-like diseases. Microarray analysis using the MDS-like bone marrow cells enabled us to identify novel targets of EZH2 in MDS tumorigenesis, including ATP-binding cassette (ABC) transporters. Derepression of Abcg2 via decreased H3K27-trimethylation was confirmed. Retroviral transduction of EZH2-dSET to MDS-like cell lines increased surface ABCG2-high populations and those cells functioned to exclude anticancer drugs as expected. Intriguingly, with Abcg2 expression alone, primary bone marrow cells could produce an MDS-like cytopenic disease in our BMT model. In our clinical specimens, ABCG2 high expressions were observed in MDS samples but not in de novo AML and CML samples. In two MDS patients, ABCG2 expression decreased along with leukemic transformation. Interestingly, two out of 33 MDS patients with extremely high expression of ABCG2 harbored the same U2AF1 mutation (Q157P). In addition, somatic mutations of EZH2 and those of either U2AF1 or SRSF2 were mutually exclusive in all investigated cases. Interestingly, U2AF1 mutants (S34F and Q157P) reduced EZH2 expression, leading the derepression of ABCG2 via decreased H3K27-trimethylation. These results indicate a link between U2AF1 mutations and ABCG2 expression via disrupted EZH2. In conclusion, different mechanisms are supposed to converge at dysregulated EZH2 in MDS. And a short form of EZH2 upregulates ABCG2 expression resulting in MDS advancing to secondary leukemia. Thus, either mutations affecting the EZH2 function or mutations of EZH2 itself could play an important role in MDS, and one of the downstream targets of EZH2 suppression in MDS pathogenesis is aberrant expression of ABCG2.

Disclosures

No relevant conflicts of interest to declare.
No Mutations of SF3B1, U2AF1, and SRSF2 spliceosomal Gene but Polymorphisms Associated with the Worse Prognosis in Multiple Myeloma
Authors: Kang Min-Gu , Kim Hye-Ran , Lee Jun Hyung , Ganapathy Thashma Pemmanda , Yang Seung-Hyeon , Hoque Moinul , Park Ra-Young , Kee Seung Jung , Kim Soo Hyun , Shin Jong-Hee , Suh Soon-Pal , Shin Myung-Geun
Published: October 2019

Recently, a striking example of the effects of acquired somatic mutations in splicing factors such as SF3B1, U2AF1, and SRSF2 has been described. The DNA sequences from abnormal blood cells of patients with hematologic malignancies has shown that a high proportion of these diseases are associated with somatic mutations of spliceosomal proteins. However, little is known about the aberrant splicing pathways in multiple myeloma (MM) patients. Therefore, this study investigated the presence and prognostic implication of splicing gene aberration in MM patients. Also, we planned functional experiments in cultured normal and Hela cell line to determine the effects of the common spliceosome mutations on cellular growth rate, cell count, and apoptosis. In total, 87 MM patients and 100 healthy controls were examined for somatic mutations in SF3B1, U2AF1, and SRSF2 by direct sequencing. Also, the clinical and laboratory characteristics of MM patients were reviewed. Regarding to the initial findings in recent reports, this study expressed the wild-type and the mutant (Q157P) U2AF1 in Hela cells and examined their proliferation.

MM patients did not show mutation in SF3B1, U2AF1,and SRSF2 splicing genes. However, the patients displayed 39198T>C or 39198T>T/C polymorphism (70.1 %) in exon 18 of SF3B1, 8345T>T/G polymorphism (13.8 %) in exon 2 of U2AF1, and 5399C>T or 5399C>C/T polymorphism (100.0 %) in exon 1 of SRSF2 (Fig. 1). In the entire cohort, the number of patients with one polymorphism, two polymorphisms, and three polymorphisms was counted up to 24.1 %, 67.8 %, and 8.0 %, respectively. Meanwhile, in 100 normal controls, polymorphism of SF3B1 exon 18 and U2AF1 exon 2 was counted up to 82.0 % and 10.0 %, respectively. Free light chain (FLC) kappa/lambda (K/L) ratio and chromosome analysis showed significant difference according to polymorphism status (p=0.002) while other clinical characteristics did not. The patient with polymorphisms in both SF3B1 and U2AF1 gene had unfavorable overall survival (p =0.067) and disease-free survival (p=0.001), compared to patients without polymorphism.

Our results show no recurrent SF3B1 and U2AF1 mutations in MM patients. Instead, polymorphisms were found in SF3B1 or U2AF1, which were significantly implicated in worse prognosis.

Figure 1. Figure 1.

Disclosures

No relevant conflicts of interest to declare.