bluuebunny/crossref_metadata_2025_split huggingface dataset:
In Vivo Selection Unmasks a Dormant Pool of Repopulating Hematopoietic Clones
Authors: Adair Jennifer E , Schefter Lauren E , Humphrys Daniel R , Haworth Kevin G , Hocum Jonah D , Trobridge Grant D. , Kiem Hans-Peter 
Published: October 2019

Abstract
Long-term clonal tracking studies utilizing hematopoietic stem and progenitor cells (HSPCs) in nonhuman primates receiving myeloablative transplantation demonstrate a successive pattern of repopulation: short-term repopulating cells are succeeded by long-term clones. However, the duration of short-term repopulation and the numbers of clones contributing to either short or long-term repopulation are unclear. Here, we tracked >11,000 unique clones in 8 pigtail macaques for up to 9 years following myeloablative transplantation with autologous, lentivirus gene-modified CD34+ HSPCs. Seven of these animals received cells expressing the P140K mutant methylguanine methyltransferase transgene, which is resistant to the combination of O6-benzylguanine (O6BG) and bis-chloroethylnitrosourea (BCNU) chemotherapy, thus conferring a selective advantage to gene-modified cells in vivo. After transplantation and before in vivo selection with O6BG/BCNU, we observed a successive pattern of hematopoietic reconstitution, with short-term clones declining within 100 days after transplantation. Within the first year after transplant, the percent of persistent clones varied from animal-to-animal, ranging from 8% to 54% of clones detected at a >1% frequency, and remained stable in the absence of selective pressure. Importantly, when animals engrafted with P140K-expressing cells were administered O6BG/BCNU we observed novel clonal patterns, which directly correlated with transplanted cell dose and time of chemotherapy administration after transplant. In all animals, chemotherapy induced emergence of previously undetected clones. In animals receiving ≤12x106 CD34+ cells/kg at the time of transplant (n = 4), chemotherapy also induced a re-emergence of previously declined short-term repopulating clones or a stabilization (i.e. decreased fluctuation) of repopulating clones identified between 100 days and 1 year after transplant. However, in animals receiving robust cell doses, ≥35x106 CD34+ cells/kg (n = 2), chemotherapy more than 1 year after transplant induced a completely novel clonal repertoire. In one animal receiving 22x106 CD34+ cells/kg at transplant, chemotherapy administration beginning <1 year (253 days) after transplant induced clonal stability, which was maintained through two additional chemotherapy treatments. These data suggest that some short-term repopulating clones may have long-term repopulation ability, but revert to a dormant phase within the first year after transplant. Additionally, these data indicate that transplant of excess repopulating cells results in early dormancy of a large proportion of repopulating clones. Together, these findings suggest that previous estimates of HSPC frequency based on clone tracking are an underestimate of true graft repopulation potential.

Disclosures
No relevant conflicts of interest to declare.
Co-Opting the Genetic Redundancy of the Pax Family to Treat PAX5 Mutated Pre-B ALL
Authors: Horwitz Marshall S , Hart Matthew R 
Published: October 2019

Abstract
Pre-B cell ALL is the most common pediatric malignancy. More than a third of cases acquire loss-of-function mutations of PAX5, a regulatory transcription factor which is required for the differentiation of B lymphocytes. Additionally, germline loss of function PAX5 mutation is a cause of familial pre-B cell ALL. These observations indicate that loss of PAX5 activity drives leukemogenesis. We have seen that PAX5 re-expression in PAX5 -mutated pre-B cell ALL restores differentiation capacity and promotes apoptosis. However, because the gene is deleted or otherwise mutated it may not be possible to easily repair or restore PAX5 expression in leukemic cells. Nevertheless, PAX5 possesses two closely related paralogs, PAX2 and PAX8, which are structurally and functionally similar to PAX5, yet are neither expressed in lymphocytes nor mutated in ALL. Toward the goal of exploiting PAX family genomic redundancy for therapeutic purposes, we have tested the ability of PAX2 and PAX8 to replace PAX5 in pre-B ALL cell lines. Preliminary results indicate that both PAX2 and PAX8 rescue B cell differentiation and function similarly to PAX5 in this context. These results include modulation of B cell developmental markers, CD10, CD19 and CD22, as well as induction of PAX5 target genes, CD79a and BACH2. Cells also display a reduction in size which may be indicative of the large to small B cell transition and importantly show increased levels of cell death following PAX2 and PAX8 expression. We have further investigated the epigenetic status of PAX2 and PAX8 promoters in B cells and find that both loci are hypermethylated, suggesting that demethylation with agents such as methotrexate, may represent a therapeutic entry point for activating expression of PAX5 paralogs. Finally, based on observations that electrical polarization during early embryogenesis regulates developmental transcription factor expression, including for PAX genes, we have tested the ability of approved drugs targeting ion channels for their ability to induce PAX factor expression and thereby complement for loss of PAX5 in pre-B cell ALL. The anti-parasitic drug ivermectin, which activates chloride channels, is one of several compounds that appear promising in early studies.

Disclosures
No relevant conflicts of interest to declare.
Induction of NK and T Cell Immune Responses Against Leukemia Cells By Bispecific NKG2D-CD16 and -CD3 Fusion Proteins
Authors: Koerner Samuel , Kanz Lothar , Grosse-Hovest Ludger , Jung Gundram , Salih Helmut R 
Published: October 2019

Abstract
Monoclonal antibodies (mAbs) have by now become an established tool in therapy of many malignancies. The interaction of a mAb's Fc portion with Fcγ receptors (FcγR) on immune effector cells is important for its efficacy, but often insufficient to potently induce antitumor immunity e.g. due to FcγR polymorphisms. Moreover, Fc parts of mAbs may bind to FcγRs expressed on non-cytotoxic cells (e.g., platelets and B cells) and interact with FcγRs that do not trigger cytotoxicity (e.g. CD16b on granulocytes). These shortcomings can be overcome by novel antibody formats like bispecific antibodies allowing for improved activation of a specific pool of effector cells. Here we report on the development and preclinical characterization of two bispecific fusion proteins that target ligands of the immunoreceptor NKG2D (NKG2DL) which are widely expressed on malignant cells but generally absent on healthy tissue. Our fusion proteins consist of the extracellular domain of NKG2D as targeting moiety for tumor-expressed NKG2DL fused to Fab-fragments of either an agonistic CD3 or CD16 antibody. Specific binding of these NKG2D-CD3 and NKG2D-CD16 constructs was confirmed using NKG2DL-transfectants with regard to their target arm and either NK cells or T cells with regard to their different effector parts. Dose titration assays revealed an increased affinity of NKG2D-CD16 to the FcγR on NK cells as compared to our previously described (e.g., Steinbacher et al, 2014) Fc-optimized NKG2D-IgG1 fusion protein, which was mirrored by a potently increased ability to induce lysis of NKG2DL-tranfectants and NKG2DL-positive primary acute myeloid leukemia (AML) cells by allogeneic NK cells. The novel NKG2D-CD3 construct in turn was found to potently activate allogeneic and autologous CD4+ and CD8+ T cells. Next we comparatively analyzed the efficacy of T cells and NK cells to lyse autologous leukemia cells upon treatment with NKG2D-CD3 and NKG2D-CD16, respectively, by using PBMC of AML patients (blast counts 30-70%) directly ex vivo in long term cytotoxicity assays. NKG2D-CD16 potently induced AML cell lysis, and this was, in line with their proliferative and higher effector potential, by far exceeded upon stimulation of T cells with NKG2D-CD3. Taken together, we here introduce novel "antibody-like" bispecific constructs that take advantage of the highly tumor-restricted expression of NKG2DL and potently activate the reactivity of NK or T cells for immunotherapy of leukemia.

Disclosures
No relevant conflicts of interest to declare.
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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.