bluuebunny/crossref_metadata_2025 huggingface dataset:
Prevalence and Clinical Correlates of JAK2 Mutations in Pediatric Down Syndrome Acute Lymphoblastic Leukemia.
Authors: Gaikwad Amos S. , Rye Cassia L. , Devidas Meenakshi , Izraeli Shai , Plon Sharon E. , Basso Giuseppe , Pession Andrea , Rabin Karen R.
Published: April 2020

Approximately three-quarters of pediatric acute lymphoblastic leukemia (ALL) cases harbor recurrent, prognostically significant chromosomal abnormalities which affect assignment of risk group and therapeutic regimen, but these abnormalities occur far less often in children with Down syndrome (DS) and ALL. Recently, novel somatic activating mutations in Janus kinase 2 (JAK2) were reported to occur uniquely in DS ALL, affecting 18% of patients (Bercovich et al, in press). Here we report the identification and clinical correlates of JAK2 mutations in an independent cohort of cases. We sequenced JAK2 exon 16 and identified mutations in 10 of 53 DS ALL cases (18.9%). All were heterozygous point mutations affecting arginine 683 (Table 1). No JAK2 mutations were identified in the 25 available paired germline DS samples, including five germline samples associated with JAK2 mutated ALL cases, indicating that mutations were somatic in these cases. Eight of the JAK2 mutations (80%) occurred in males (p<0.03), whereas the full cohort contained 24 males (45%). No significant correlation was observed between JAK2 mutation status and age, initial white blood count (WBC), or 5-year event-free survival or overall survival. Our results confirm the 18% prevalence of JAK2 mutations at arginine 683 in DS ALL, but differ from the prior report in finding a male predominance of JAK2 mutations, and no association with age or initial WBC, differences which may be attributable to sample size. These results confirm that activation of the JAK-STAT pathway via a specific JAK2 mutation occurs in approximately one-fifth of cases of DS ALL.

Sequence Amino acid Cases (n) AGA (wild-type) Arginine 43 GGA Glycine 6 AGT Serine 2 AGC Serine 1 ACA Threonine 1

Table 1. JAK2 mutation status in pediatric DS ALL
MRD Monitoring of Childhood ALL Using Hierarchical Clustering and Support Vector Machine Learning of Complex Multi-Parameter Flow Cytometry Data.
Authors: Fiser Karel , Sieger Tomas , Schumich Angela , Irving Julie , Dworzak Michael N , Vormoor Josef
Published: April 2020

Flow cytometry is an important tool both for research and diagnostics of hematologic malignancies - including monitoring of minimal residual disease (MRD). Recent progress and more widespread availability of 6 and higher color flow cytometry leads to complex, information-rich datasets which are very challenging to analyze. Here, we validate a novel approach to multi-parameter flow data analysis in MRD flow data sets from 39 ALL patients (including 23 patients from the I-BFM list mode data (LMD) ring trial). The approach combines hierarchical clustering (HCA) using a newly developed algorithm and support vector machine (SVM) learning. The algorithm employs a scale-invariant Mahalanobis distance measurement for merging clusters. This reflects the extended ellipsoid shape of the populations and is better suited for flow cytometric data compared with standard HCA metrics. The resulting hierarchical tree, combined with the heatmap of the CD marker expression allows visualization of hierarchically clustered data of all analyzed parameters displayed in a single plot. The clusters from HCA (representing the ALL blast population at diagnosis) were used to train SVM classifiers which were then applied to test for presence of a matching population in the test sample (follow-up sample). All work was carried out in MATLAB (MathWorks, Inc.). Using HCA, we have been able to detect the leukemic blast population in diagnostic and follow-up datasets (n=81) from three centers. The correlation (Pearson correlation coefficient = 0.98) between HCA and the standard gating approach was highly comparable to inter-laboratory comparisons within the I-BFM LMD ring trial (Dworzak MN et al; Cytometry B Clin Cytom. 2008 Jun 11.). To further improve sensitivity and exact quantification of low MRD levels and to automate MRD detection, we combined HCA with SVM learning. We have analyzed 21 samples from 5 patients with MRD levels between 0.004 to 57.54%. HCA plus SVM correlated better with standard gating results than HCA alone in particular in samples with low MRD levels (<10−3). In summary, HCA in combination with SVM proved to be a strong analytical tool for flow cytometry with the potential for automated MRD detection. We validated this approach for use in ALL diagnostics and MRD monitoring by comparison with expert-based gating analyses of I-BFM LMD ring trial data.
The Leukemia-Associated Antigen PRAME Is Overexpressed in Myeloid Leukemias and Inhibits Cell Differentiation by Blocking the Receptor for Retinoic Acid (RAR)-Signaling in Vitro and Is Therefore a Interesting Candidate for Targeted Immunotherapies.
Authors: Greiner Jochen , Bullinger Lars , Giannopoulos Krzysztof , Schmitt Anita , Goetz Marlies , Kienle Lena , Döhner Hartmut , Schmitt Michael
Published: April 2020

The leukemia-associated antigen PRAME (preferentially expressed antigen of melanoma) is frequently expressed in several solid tumors. Earlier, we reported (Greiner et al., Blood 2006) that co-expression of leukemia-associated antigens including PRAME constituted a favorable prognostic parameter for AML patients. By microassay analysis and conventional RT-PCR, we detected over-expression of PRAME in more than 60% of AML and of CML patients. In contrast, PRAME is expressed neither in normal CD34-positive hematopoietic stem cells nor in normal tissues (other than testis and placenta). Here, we describe for the first time that retinoic acid-induced cell proliferation and differentiation of several AML cell lines is dependent on PRAME expression: PRAME negative cell lines treated with all-trans retinoic acid (ATRA) in cell culture showed lower cell proliferation than PRAME positive cells as assessed by cell counts and FACS analysis for BrdU, but an increase of cell differentiation detected by FACS analysis for CD66b in contrast with PRAME positive leukemia cell lines. The leukemia-associated antigen PRAME seems to be responsible for the resistance of PRAME-positive AML to ATRA treatment. We detected no differences in induction of apoptosis in PRAME-positive or PRAME-negative cell lines treated with ATRA in FACS analysis for annexin. Over-expression of the antigen and this critical role of PRAME in cell differentiation might be the reason for specific T cell induction against PRAME-derived peptides. To detect specific T cell responses against PRAME-derived peptides we examined samples from healthy volunteers and AML patients. Positive results in ELISPOT assays for IFN-g and Granzyme B secretion were observed in 70% of AML cases analyzed for the peptide PRAME-P3. In chromium release assays, we found a PRAME-specific cell lysis of PRAME-positive AML blasts. Taken together, PRAME is involved in a crucial mechanism for cell growth of leukemic cells, induces specific T cell responses in a high frequency of AML patients, and this constitutes an appropriate target structure for specific immunotherapies or other targeted therapies in AML.
The Critical Role of Iκbα Dependent Nuclear Export of NF-κb in B-Cell Development.
Authors: Yang David T , Wuerzberger-Davis Shelly , Chen Yuhong , Yu Mei , Zeng Hu , Bates Paul , Wen Renren , Wang Demin , Miyamoto Shigeki
Published: April 2020

Activity of the nuclear factor-κB (NF-κB) family of transcription factors is tightly regulated by its inhibitor, IκBα, through cytoplasmic localization of latent NF-κB: IκBα complexes. This arrangement is essential for efficient signal-inducible activation and regulation of biologic functions. Maintenance of cytoplasmic localization of latent NF-κB: IκBα complex requires continuous nuclear export that is dependent on the N-terminal nuclear export sequence (N-NES) of IκBα. While these mechanisms have been elucidated through in vitro studies, the biological significance of this “nucleocytoplasmic shuttling” has yet to be evaluated in vivo. To address this, we derived mice harboring germ-line M45A, L48A, and I52A amino acid substitutions in the N-NES of IκBα. In splenic B-cells, the disrupted N-NES caused constitutive nuclear accumulation of IκBα and inactive c-Rel containing complexes but surprisingly not IκBα: p65 complexes. Since p65 contains a NES sequence and c-Rel does not, nuclear export of N-NES mutant IκBα:NF-κB complexes appear to be NF-κB family member dependent. Functionally, NF-κB activity in splenic B-cells after stimulation with IgM or LPS was clearly reduced in the mutants compared to wild-type by electrophoretic mobility shift assay. B-cell development in the bone marrow of mice harboring the mutation was impaired, showing a preponderance of pro/pre B-cells and few mature B-cells compared to their wild type littermates (p < 0.001). Concordantly, there were significantly fewer B-cells in the spleen (p < 0.05) and lymph nodes (p < 0.01) of the mutant mice. Additionally, populations of T2, follicular (FO), and marginal zone (MZ) B-cells, which represent mature B-cells in the spleen, were also reduced in the mutant mice (p < 0.001). To demonstrate that this B-cell maturation defect in IκBα mutant mice was B-cell intrinsic, sublethally irradiated Jak3-deficient mice were transplanted with BM from either wild-type or mutant mice. B-cell development in mice transplanted with mutant donors was impaired relative to those with wild-type donors in a fashion identical to that of the primary mutants described above. Finally, severe phenotypes in inguinal lymph nodes and Peyer’s patch development were present, with mutant mice frequently lacking these secondary organs/tissues, the underlying mechanisms of which are currently being investigated. In conclusion, our findings uncover an in vivo mechanism controlling NF-κB localization and its essential role in the generation of mature B-cells and certain secondary lymphoid organs.
Suppression of Allogeneic Immune Responses by Human TCRαβ+ CD4− CD8− Double-Negative T Cells.
Authors: Voelkl Simon , Gary Regina AM , Mackensen Andreas
Published: April 2020

Regulatory T lymphocytes play an important role in the maintenance of immune tolerance to self antigens and are involved in downregulating immune responses in autoimmunity, transplant rejection and tumor immunity. Numerous studies have demonstrated the existence of distinct T cell subsets with immunoregulatory properties. Recently, a novel subset of TCRαβ+ CD4− CD8− (double-negative, DN) T cells has been characterized to specifically suppress immune responses in both mice and humans. Here we demonstrate for the first time that human DN T cells are highly potent suppressor cells of allogeneic CD4+ or CD8+ T cell responses after priming with allogeneic antigen presenting cells (APC). A prerequisite for the immunosuppressive activity is the repetitive priming with allogeneic dendritic cells whereas stimulation with artificial APCs has no effect. Using a transwell system we could show that the suppressive activity against allogeneic immune responses, mediated by DN T cells, requires cell contact. In contrast to murine DN T cells, which eliminate effector T cells via a fas/fasL or perforin/granzyme pathway, human DN T cells suppress the proliferation of alloreactive T cells in an active manner. Taken together, our data indicate that human DN T cells possess strong immunosuppressive effects on alloreactive CD4+ or CD8+ T cells. DN T cells may serve to limit clonal expansion of alloantigen-specific T cells after allogeneic peripheral stem cell transplantation.
T Cells Demonstrate Enhanced Specificity for CD19+ Malignancies When Stimulated with IL-21.
Authors: Singh Harjeet , Huls Mary Helen , Dawson Margaret J. , Mi Tiejuan , Dotti Gianpietro , Lee Dean A. , Kebriaei Partow , Shpall Elizabeth J. , Hackett Perry B. , June Carl H. , Champlin Richard E. , Cooper Laurence J.N.
Published: April 2020

T cells genetically modified to express CD19-specific chimeric antigen receptors (CARs) are being evaluated in early-phase clinical trials in patients with B-lineage malignancies. Therapeutic success is predicted by ability of infused CAR+ T cells to both persist and kill in an antigen-dependent fashion. The first of these dual goals can be achieved by altering the CAR molecule to provide T-cell survival signals through a chimeric CD28 endodomain (designated CD19RCD28). We now report that altering the culturing microenvironment with IL-21 improves antigen-dependent cytolysis of T cells when propagated on CD19+ artificial antigen presenting cells (aAPC) derived from K562. To test whether IL-21 acts in conjunction with CD28 signaling to support acquisition of redirected effector functions we electro-transferred quiescent T cells from peripheral blood with Sleeping Beauty system DNA plasmids to introduce CD19RCD28 CAR transposon. Selective outgrowth of CAR+ T cells was achieved on CD19+ aAPC that provide co-stimulation with the addition of exogenous IL-2 and/or IL-21. When IL-21 was present there was preferential numeric expansion of CD19-specific CD8+ T cells which lysed and produced IFN-g in response to CD19 (Figure). Furthermore, the CD8+CAR+ T cells displayed a central memory (CM) cell surface phenotype characterized as CD62L+ and CD28+. In contrast, genetically modified T cells propagated with exogenous IL-2 resulted in predominately CD19-specific CM CD4+ T cells. Thus, cytokines can be used to tailor the CD8/CD4 ratio of CAR+ T cells derived from peripheral blood. These data demonstrate that the dual goals of persistence and lysis can be achieved by altering CAR and the cytokine milieu and have implications for infusing CAR+ T cells in next-generation immunotherapy trials.

Figure Figure
BCX-4208 (RO5092888), a Purine Nucleoside Phosphorylase (PNP) Inhibitor, Is a Novel, Potent Orally Active Anti-T-Cell and B-Cell Agent.
Authors: Bantia Shanta , Parker Cynthia , Upshaw Ramanda , Kilpatrick John Michael , Cunningham Amanda , Zhang Jianwen , Kotian Pravin , Morris Philip , Chand Pooran , Babu Yarlagadda S.
Published: April 2020

The profound suppression of T-cell immunity seen in patients with an inherited PNP deficiency supports the potential application of inhibitors of this purine salvage enzyme in the therapy of T-cell malignancies and T-cell mediated autoimmune diseases. About thirty percent of PNP deficient patients also show evidence of B-cell dysfunction. BCX-4208 is a novel potent transition state analog inhibitor of PNP (IC50 ~ 0.0005 μM) and in the presence of 10 μM deoxyguanosine (dGuo), inhibits human lymphocyte proliferation induced by MLR, IL-2 or Con-A with IC50s of 0.159, 0.26 and 0.73 μM, respectively. The IC50 for dGuo in the same assays in the presence of 1 μM BCX-4208 ranges from 1–3 μM. Neither BCX-4208 alone nor dGuo alone inhibits proliferation of lymphocytes. In the presence of PNP inhibitor, dGuo is converted to dGMP and then to dGTP. Accumulation of dGTP results in the alteration of deoxynucleotide (dNTP) pools, causing death of cells via a mechanism characteristic of apoptosis. In vitro data demonstrates that following exposure to BCX-4208 and dGuo, dGTP in human lymphocytes is elevated and a 5–8 fold increase in dGTP results in 50% inhibition of lymphocyte proliferation. Flow cytometric analyses of human lymphocytes using annexin-V staining reveal that BCX-4208 in the presence of dGuo induces cellular apoptosis not only in T cells (CD3+), but also in B cells (CD20+; CD19+) (Table 1). BCX-4208 is orally bioavailable in mice, can achieve maximal inhibition of PNP, and elevates plasma dGuo levels to 3–5 μM (predose levels < 0.004 μM), which is similar to levels seen in PNP deficient patients and to levels needed to cause apoptosis in T and B-cells. These data support the evaluation of BCX-4208 in the treatment of not only T-cell mediated diseases but also B-cell mediated diseases. BCX- 4208 is currently undergoing early clinical investigation in patients with psoriasis.

Table 1.

Cell subsets % Apoptotic cells mean ± SEM (n = 4–7) Vehicle Treatment group *p ≤ 0.01 compared to vehicle CD3+ 7.2 ± 0.9 18.3 ± 3.7* CD20+ 24.0 ± 3.6 41.8 ± 6.2* CD19+ 25.0 ± 3.1 51.6 ± 7.4*
Role of Human NK Cells in Dentric-Cell Based Vaccine.
Authors: Bos Gerard M.J. , Van Elssen Janine CHMJ , Vanderlocht Joris , Senden-Gijsbers Brigitte LMG , Germeraad Wilfred LMG
Published: April 2020

Figure Figure

Besides their prominent role in the destruction of altered self-cells, natural killer (NK) cells have been shown to potentiate T cell responses by interacting with dendritic cells (DC). In mouse models as well as in a recent human study it has been demonstrated that DC might activate NK cells. In the context of dendritic cell-based vaccines – i.e. optimising the optimal maturation cocktail - it remains to be determined if and how NK-DC interactions depend on differential DC maturation and what factors influence the NK activation.. By comparing differential DC differentiation (IL-4/GM-CSF and IL-13/GMCSF) and maturation cocktails (IFN-γ/FMKp and PGE2/TNF-α), we show that the ability of human DCs to attract NK cells is imprinted during DC maturation. Only FMKP/IFN-γ (stimulation Toll like receptor 2 and 4) maturated DCs have the capacity to actively recruit NK cells in vitro and our data indicate that CCR5 is the dominant chemokine receptor in this recruitment (Figure 1). Furthermore, in contrast to PGE2/TNF-α matured DC, FMKP/IFN-γ maturated DCs activate NK cells to produce IFN-γ in a IL-12/IL18 dependent manner, of which we show it contributes to strong TH1 polarization. In addition upon contact with these DCs NK cells upregulate their lymph node homing receptors, possibly inducing secondary migration to the lymph nodes.

In conclusion, besides the identification of a superior DC maturation cocktail which contributes to NK-DC interactions, we identified a novel recruitment mechanism for peripheral human NK cells which may contribute to secondary, central DC-NK interactions and strong TH1 polarization.
IL-10 Present in the B Lymphoma Microenvironment Drives Macrophages to Efficient Tumour Suppressing Cells with High Phagocytic Activity in Presence of Rituximab.
Authors: Golay Josee , Leidi Marzia , Gotti Elisa , Palumbo Giuseppe , Introna Martino
Published: April 2020

Since macrophages have been implicated as major players in the mechanism of action of Rituximab (Mabthera®) in vivo, we have investigated the factors that modulate their tumour cell killing potential in vitro. Human macrophages expressing CD16, CD32 and CD64, were differentiated from CD14+ peripheral blood monocytes by culture for 5–7 days in presence of M-CSF. Binding of rituximab opsonised target cells was measured after 5 minutes incubation of macrophages with CFSE labelled B-CLL cells and FACS analysis. Phagocytosis was quantified after 2 hours at 37°C by microscopic count of stained slide preparations. ADCC was measured by standard release assays. Rituximab induced specific binding of CD20+ target cells to macrophages and this was followed by phagocytosis, but not ADCC. Phagocytosis was maximal at 0.1 μg/ml rituximab and was not significantly affected by CD20 expression levels on target cells. The CD16A polymorphism at amino acid 158 (Val/Phe) that affects IgG binding did not significantly modify the efficacy of phagocytosis at different rituximab doses, possibly due to the role of additional Fcγ receptors. Indeed phagocytosis was blocked by excess human immunoglobulins. Since macrophages can be differentiated to M1 type or M2 type cells with either GM-CSF or M-CSF, respectively, and can be classically activated by pro-inflammatory stimuli (IFNγ + LPS) or undergo alternative activation with cytokines such as IL-4 or IL-10, we have analysed the effect of these different polarisation programs on the phagocytosis mediated by rituximab. Macrophages differentiated in presence of M-CSF showed a 2–3 fold greater phagocytic capacity compared to GM-CSF induced cells. Furthermore addition of IL-10 significantly increased, whereas IL-4 decreased phagocytosis by both M-CSF and GM-CSF differentiated macrophages. LPS/IFNγ had little effect. Expression of CD16, CD32A/C and CD64 correlated with the phagocytic capacity of the different polarised populations, suggesting that M-CSF and IL-10 induced maximal phagocytosis through upregulation of several activating FcγRs. Several B lymphoma cell lines were observed to secrete 400–1300 pg/ml IL-10 in vitro and co-culture of human macrophages with lymphoma supernatant increased significantly their phagocytic capacity. These data suggest that IL-10 produced in the lymphoma microenviroment may activate macrophages to become M2 type cells with high phagocytic capacity. Thus usually tumour promoting M2 type macrophages within the lymphoma would become tumour suppressing in presence of rituximab.
Combined Treatment with Short Synthetic Anti-Lymphoma Peptide and CpG ODN; A Therapeutic Vaccine Which cures the A20 Lymphoma in Balb/C Mice.
Authors: Kolstad Arne , Houot Roch , Berge Gerd , Rekdal Øystein , Czerwinski Debra , Goldstein Matthew , Brody Joshua , Levy R.
Published: April 2020

Background. The short synthetic peptide 302 has been shown to induce rapid membrane disruption of lymphoma cells in vitro and necrosis of local tumors in the A20 lymphoma model in Balb/c mice. In order to stimulate the immune system to generate an anti-tumor response we designed a model where intra-tumor injections of peptide 302 was combined with sc injections of the Toll-like receptor 9 binding synthetic oligonucleotide CpG 1826.

Methods. Balb/c mice were inoculated with A20 lymphoma cells sub-cutaneously (s.c.) on the abdomen. When the tumors reached a size of 5–7 mm, 302 peptide was administered directly into the tumors on days 1 and 6. CpG 1826 was injected s.c. on days 1–4 and 6–8. Tumor growth was measured repeatedly during follow-up. A20-specific T-cell responses were detected by culturing peripheral blood lymphocytes from treated animals for 24 hours with A20 lymphoma cells and analyzing for intracellular IFN-γ production by flow cytometry. To dissect the role of T-cell subtypes, the treatment was performed in animals depleted for CD4 or CD8 positive T-cells. In order to show A20 specific immunological memory, cured animals were re-challenged with the A20 lymphoma or the carcinoma cell line CT26.

Results. Combined treatment with peptide 302 and CpG 1826 cured 8 out of 10 mice, compared to only 2 out of 10 mice who received peptide 302 alone or CpG 1826 alone. Cured mice were followed for 9 weeks without relapsing. Similar results with the combination of peptide 302 and CpG were observed in a separate experiment. The highest cure rate was achieved when injecting CpG 1826 s.c. in the tumors draining lymph node area as compared to administration of CPG 1826 in a non-draining lymph node region or intra-peritoneal. Animals treated with peptide 302 + CpG 1826 or CpG 1826 alone developed CD8-specific IFN-γ responses against A20 cells. In one separate experiment CD8 knock-out mice did not respond to the treatment, unlike animals depleted for CD4+ cells and normal mice. Only 1 out of 10 cured animals re-challenged with the A20 lymphoma developed a new tumor, a result that was reproduced in a second experiment.

Conclusion: Treatment with intra-tumor injections of the anti-lymphoma peptide 302 in combination with CpG 1826 s.c. in the draining lymph node region cured established A20 tumors, induced tumor-specific CD8 positive tumor-reactive T-cells, and induced specific immunological memory. This principle represents a novel therapeutic vaccine approach.
Inhibition of Human Lymphoma Cell Growth by the PKC-Beta Selective Inhibitor Enzastaurin (LY317615) in Combination with Multiple Therapeutic Agents.
Authors: Rossi Randall M , Balys Marlene , Franklin Dean , Grose Valerie , Fisher Richard I , Jordan Craig
Published: April 2020

Previous studies in our lab have shown that the PKC-beta inhibitor, enzastaurin (LY317615), when used to treat a panel of human diffuse large cell lymphoma (DLCL) lines, was able to induce cell death in vitro and substantially reduce tumor growth in xenograft assays. These findings support the hypothesis that activation of PKC contributes to tumor cell survival and proliferation, which has been implicated in the pathogenesis of human B cell lymphomas. Specifically, PKC-beta activation is increased in tumor cells from patients with poor prognosis DLCL, suggesting that PKC-beta may be a target for therapeutic intervention. In the present study, we have explored the interaction of enzastaurin with a panel of well characterized therapeutic agents to evaluate whether its anti-tumor activity can potentially be enhanced. Drugs were chosen for analysis based either on known single agent activity in lymphoma, or by preclinical evaluation indicating potential synergy with enzastaurin. For in vitro culture assays (48–72 hr treatment), the addition of gemcitabine, rapamycin, or bortezomib, increased the cytotoxicity of enzastaurin from 2 to 7 fold. This effect was evident with multiple human DLCL cell lines, (OCI-Ly3, 7, 10, 19, and SUDHL-4, and 6), as well as two independent primary DLCL cultures. For in vivo studies, subcutaneous transplantation of the DLCL cell line OCI-Ly19, (previously engineered to express luciferase which allows for real time in vivo imaging), or a primary DLCL isolate, into immune deficient NOD/SCID mice formed reproducible tumors. Recipient animals were separated into uniform cohorts when the tumors were of <=500 cubic mm in size. The animals were then simultaneously or sequentially treated with enzastaurin, (150 mg/kg b.i.d. via oral gavage) and a secondary drug, either gemcitabine, (2.5 or 5.0 mg/kg 1x/3 days IP), bortezomib, (0.4 mg/kg twice weekly IP), rapamycin, (1.0 mg/kg, daily IP), or rituxan, (5 mg/kg, weekly IP). Imaging and analysis of tumor volumes showed that addition of either rituxan or rapamycin provided no additional benefit in comparison to enzastaurin alone during the course of treatment. In contrast, the combination of either gemcitabine or bortezomib with enzastaurin demonstrated significantly reduced tumor volumes in comparison to enzastaurin alone (17% to 38% greater decrease with enzastaurin + gemcitabine, and 50% greater decrease in tumor volume with enzastaurin + bortezomib). These data suggest that the use of enzastaurin in combination with existing therapeutic drugs (gemcitabine or bortezomib) has the potential to limit tumor size/growth while lowering dose levels and thereby reducing potential side effects associated with traditional treatments.
Pharicin B, a Novel Diterpenoid, Stabilizes Retinoic Acid Receptor-α Protein and Induces Differentiation of AML Cells in the Presence of Physiological Doses of ATRA.
Authors: Chen Guo-Qiang , Gu Zhi-Min , Zhou Mei-Yi , Wu Ying-Li , Huang Ying
Published: April 2020

Retinoids, a generic term that covers compounds including both naturally dietary vitamin A (retinol) metabolites and active synthetic analogs, exert their pleiotropic effects such as anticancer activity through the three retinoic acid receptors (RARs) subtypes [RARα, RARβ and RARγ]. The most impressive example of retinoid anticancer activity is the successful application of all-trans retinoic acid (ATRA) in the treatment of patients with acute promyelocytic leukemia (APL), a unique subtype of acute myelogenous leukemia (AML) which characterized with the specific reciprocal chromosome translocation t(15;17) that results in the expression of leukemia-promoting promyelocytic leukemia-retinoic acid receptor-α (PML-RARα) chimeric protein. However, retinoid resistance frequently occurred in ATRA-treated patients. Isodon xerophilus, a perennial shrub native to Southern China, has been used as an anti-tumor, anti-inflammatory, and anti-microbial agent in Chinese herb medicine for a long history. During the past 30 years, a large number of ent-kauranoids have been isolated from the genus Isodon, many of which exhibit potent antitumor activities with a relatively low toxicity. In this work, we identified a novel ent-kaurene diterpenoid named pharicin B to rapidly stabilize RARα as well as PML-RARα protein in AML cell lines. More intriguingly, it also antagonizes ATRA-induced degradation of RARα and PML-RARα proteins. The interesting finding promotes us to investigate its possible effects on AML cells. Our results demonstrated that pharicin B at nontoxic concentration suppresses growth in APL cell line NB4 and myeloblactic leukemic U937 and THP-1 cell lines. Together with exceedingly low concentration of ATRA and RARα specific agonist AM580 existed, pharicin B significantly triggered all the three cell lines and some NB4-derived ATRA-resistant cell lines such as NB4-MR2 and NB4-LR1 (but not NB4-LR2) to undergo myeloid maturation, as evidenced by morphology, CD11/CD14 expression and NBT reduction test. All these results proposed that pharicin B would be a good tool for investigating mechanisms of RARα stabilization and degradation induced by ATRA as well as retinoid resistance, and its combination with ATRA might present the clinical potentials for differentiation-inducing therapy of APL and other AML patients.
Identification of p53 Aberration-Dependent as Well as Non-p53-Mediated Resistance to MDM2 Inhibitors in Acute Myelogenous Leukemia.
Authors: Long Jianting , Ouillette Peter , Parkin Brian , Erba Harry , Wang Shaomeng , Malek Sami
Published: April 2020

MDM2 inhibitors are under development as novel therapeutic agents in cancers and multiple such compounds have entered early-phase clinical trials. AML is characterized by a relatively low incidence of p53 mutations, thus constituting a tumor type that may have high response rates to MDM2 inhibitors.

We have measured apoptosis induction following ex-vivo treatment with the MDM2 inhibitors MI63 and MI-219 in highly purified AML blasts from 80 patients using Annexin-V FACS-based readouts at 40 hours post treatment. IC50 values for apoptosis induction were calculated using dose response curves and the program XLfit. Data were integrated with p53 exons 5–9 sequence data and p53 immunoblotting data before and after MDM2 inhibitor treatment.

Results: AML blast sensitivities to treatment with MI219 fell into three classes: sensitive with IC50 values <2μM (22/80=28% of all cases), partial resistance with IC50 values ≥2 to <10μM (32/80=40% of all cases) and resistant with IC50 >10μM (26/80=32% of all cases). All eight cases with p53 exons 5–9 sequence mutations were resistant to MI-219. p53 immunoblotting analysis before and after MI219 treatment revealed that the vast majority of resistant cases with wild-type p53 exons 5–9 displayed wild-type p53 induction.

Conclusion: AML is characterized by a substantial proportion of cases with partial or complete resistance to MDM2 inhibitor treatment. Further, given that the majority of these cases display wild-type p53 induction after MDM2 inhibitor treatment, it can be concluded that post-p53 network defects confer blocks to apoptosis induction in resistant AML blasts. To identify and catalogue these defects we have begun expression array-based analysis of transcriptome differences in sensitive versus resistant cases, SNP 6.0 array-based analyses of genomic aberrations in sensitive versus resistant cases and targeted molecule interrogations.

In summary, our data provide important supportive information regarding the planned initiation of human clinical trials with MDM2 inhibitors in AML. Such trials may benefit from ex-vivo testing of AML blasts for sensitivity to MDM2 inhibitors as part of pre-enrollment patient selection.
Analysis of Aurora B Kinase in Non-Hodgkin’s Lymphoma.
Authors: Ikezoe Takayuki , Takeuchi Tamotsu , Nishioka Chie , Adachi Yoshihiro , Furihata Mutsuo , Koeffler H. Phillip , Yokoyama Akihito
Published: April 2020

Aurora B kinase is one of the key regulators of mitosis and has been reported to be over-expressed in a various malignancies including acute myelogenous leukemia; however, expression of Aurora B in non-Hodgkin’s lymphoma (NHL) and its role in lymphoma genesis remain to be fully elucidated. This study explored the levels of Aurora B in seventy four lymph nodes and tumor specimens excised operatively from individuals with various types of NHLs including diffuse large B cell lymphoma (n=22, DLBCL), follicular lymphoma (n=12, FL), Burkitt’s lymphoma (n=10, BL), mantle cell lymphoma (n=4, MCL), mucosa associated lymphoid tissue (n=4, MALT), adult T-cell leukemia/lymphoma (n=11, ATL), peripheral T-cell lymphoma (n=8, PTCL), and Hodgkin’s lymphoma (n=3, HL). Immunohistochemical examination found that DLBCL (11/22, 50 %) and BL (8/10, 80 %) cells highly (percentage of positive cells, >20 %) expressed Aurora B in their nucleus. On the other hand, FL (11/12, 92 %), MCL (1/4, 25 %), and MALT (1/4, 25 %) cells slightly (percentage of positive cells, 5–20 %) expressed Aurora B and none of them, except for one case of FL, highly expressed this protein kinase, suggesting that expression of Aurora B correlated with histological grade in B cell NHLs (p=0.0004). We next examined whether Aurora B could be a molecular target for treatment of NHLs utilizing BALM-14, -18, and -27, Daudi, Ramos, and Raji Burkitt’s lymphoma/leukemia cells. Exposure of these cells to AZD1152, a selective inhibitor of Aurora B kinase, potently induced apoptosis in association with activation of caspase 3, as measured by annexin V staining and Western blot analysis, respectively. Moreover, we found that AZD1152 synergistically enhanced the effects of vincristine (VCR), a tubulin depolymerizing agent, to induce growth arrest and apoptosis of these cells. Further experiments found that VCR increased levels of the phosphorylated forms of Aurora B (p-Aurora B) via activation of c-Jun N-terminal kinase (JNK), which was blocked in the presence of AZD1152 in BALM-27 cells. Taken together, Aurora B kinase is a promising molecular target for treatment of individuals with aggressive types of NHLs. The combined administration of AZD1152 and the conventional chemotherapeutic agents to patients with NHL warrants further investigation.
Hedgehog Inhibitors Reduce the Survival and Drug Resistance in CD34+ Leukemic Cells.
Authors: Kobune Masayoshi , Murase Kazuyuki , Iyama Satoshi , Sato Tsutomu , Kawano Yutaka , Kikuchi Shohei , Miyanishi Koji , Takimoto Rishu , Kato Junji
Published: April 2020

Aberrant reactivation of Hedgehog (Hh) signaling has been described in a wide variety of human cancers and its role in maintenance of self-renewal of cancer stem cells. We previously showed that Indian Hh (Ihh) and its receptor molecules, Patched and Smoothened are expressed in cord blood (CB) CD34+ cells and Ihh regulate proliferation of hematopoietic myeloid- and lymphoid-repopulating cells in vivo. However, the involvement of Hh signaling in proliferation of leukemic cells has remained unclear. In this study, we assessed the possibility that Hh pathway activation contributes to the growth, survival and drug resistance of acute myeloid leukemic (AML) cells. Hh signaling in leukemic cell lines and bone marrow (BM) CD34+ cells were screened by RT-PCR and a Hh signaling reporter assay. We have found that Ihh were expressed in BM CD34+ cells and most of human AML cell lines, HL60, U937, KG1, Kasumi-1, Kasumi3 and TF-1. In contrast, Hh receptor components, Patched and Smoothened, were detected in BM CD34+ cells and cytokine responsible CD34+ AML cell lines such as Kasumi-1, Kasumi-3 and TF-1. Moreover, the downstream transcription factor GLI1 or GLI2 were expressed in BM CD34+ cells and these CD34+ cell lines, indicating that Hh signaling was active in these cells. We further assessed whether Hh signaling transmit to GLI1 using GLI1-responsive luciferase assay. GLI-responsive reporter plasmid (TK-6GBS-Luc) was transduced into these cells in the presence or absence of anti-Hh neutralizing antibody 5E1. TK-6GBS-Luc-transduced Kasumi-1, Kasumi-3 and TF-1 cells showed high luciferase activity. Furthermore, the luciferase activity of these cells was significantly reduced in the presence of 10 μg/ml anti-Hh neutralizing antibody. These results clearly indicate that Ihh signaling transmits into these CD34+ leukemic cell lines in autocrine manner. We next examined the effect of Hh inhibitors on these CD34+ leukemic cell lines. Inhibition of Hh signaling with the naturally derived chemical Smoothened antagonist Cyclopamine, endogenous Hh inhibitor Hedgehog-interacting protein or anti-Hedgehog neutralizing antibody reduces the entry of cell cycle of CD34+ cell lines and eventually induces apoptosis in these cells after 48 hr exposure although these CD34+ cell lines exhibit resistance to cytarabine (Ara-C) exposure. In contrast, cyclopamine did not affect growth and survival of the U937 or HL-60 cell line that lacks expression of Hh receptor components. Furthermore, combination of 10 μM cyclopamine significantly reduced the drug resistance against Ara-C in CD34+ cell lines. These results suggest that Hh pathway activation is a feature of CD34+ myeloid leukemic cells and inhibition of Hh signaling pathway may be a therapeutic strategy in the treatment of AML.
Identification of Heat Shock Protein 32 (Hsp32) as a Novel Target in Acute Lymphoblastic Leukemia (ALL).
Authors: Meyer Renata A. , Herrmann Harald , Gleixner Karoline V. , Gruze Alexander , Cerny-Reiterer Sabine , Hubmann Rainer , Shehata Medhat , Mayerhofer Matthias , Sperr Wolfgang R. , Pickl Winfried F. , Melo Junia V. , Bharate Gahininath Y. , Jäger Ulrich , Maeda Hiroshi , Valent Peter
Published: April 2020

Heat shock proteins (Hsp) are increasingly employed as therapeutic targets in various solid tumors and leukemias. We have recently shown that Hsp32 is expressed in leukemic cells and serves as a survival-factor and molecular target in Ph+ chronic myeloid leukemia. In the present study, we examined the expression and functional role of Hsp32 in acute lymphoblastic leukemia (ALL). Leukemic cells were obtained from patients with Ph+ ALL (n=5) and Ph− ALL (n=5). In addition, Ph+ ALL cell lines (Z-119, BV-173, TOM-1, NALM-1) and Ph− ALL cell lines (RAJI, RAMOS, REH, BL-41) were used. As assessed by immunocytochemistry and qRT-PCR, leukemic cells were found to express the Hsp32 protein as well as Hsp32 mRNA in all patients and in all cell lines examined. The Hsp32-inductor hemin was found to promote the expression of Hsp32 in leukemic cells. To determine the functional role of Hsp32 in lymphoblasts, an siRNA against Hsp32 was applied. The siRNA-induced knock down of Hsp32 in RAJI cells was found to be associated with reduced growth and with an increase in apoptotic cells compared to a control siRNA against luciferase (p<0.05). In a next step, two pharmacologic inhibitors of Hsp32, pegylated zinc protoporphyrine (PEG-ZnPP) and styrene maleic acid-micelle-encapsulated ZnPP (SMA-ZnPP), were applied. As assessed by 3H-thymidine uptake experiments, both drugs were found to inhibit proliferation in the BCR/ABL+ cell lines Z-119, BV-173, and TOM-1, and in the BCR/ABL-negative ALL cell lines RAJI, RAMOS, REH, and BL-41. The effects of PEG-ZnPP and SMA-ZnPP were dose-dependent with IC50 values ranging between 1 and 10 μM, and were found to be associated with apoptosis as determined by microscopy as well as by flow cytometry and AnnexinV-staining. In NALM-1 cells, PEG-ZnPP and SMA-ZnPP also produced apoptosis and growth arrest, but the IC50 for SMA-ZnPP was slightly higher compared to other cell lines (20 μM). Effects of Hsp32-targeting drugs were also observed in primary leukemic cells obtained from patients with Ph+ ALL and Ph− ALL, with IC50 values ranging between 1 and 10 μM. No major differences were found when comparing results in imatinib-sensitive and imatinib-resistant patients. In drug combination experiments, Hsp32-targeting drugs were found to cooperate with imatinib and with AMN107 (nilotinib) in producing growth-inhibition and apoptosis in all Ph+ ALL cell lines tested. Furthermore, we were able to demonstrate strong cooperative antileukemic effects when applying Hsp32-targeting drugs in combination with bendamustine. Overall, these results suggest that Hsp32 may be a novel molecular target in ALL.
Targeting the Mnk Pathway Enhances Chemotherapy-Induced Antileukemic Responses.
Authors: Altman Jessica K , Glaser Heather , Redig Amanda , Sassano Antonella , Tallman Martin S , Platanias Leonidas C
Published: April 2020

Mnk kinases (mitogen-activated protein kinase [MAPK]-interacting kinases) are downstream effectors of Map kinase pathways, including the MEK/Erk and the p38 Map kinase signaling cascades. We have previously shown that Mnk kinases and the p38 Map kinase pathway are activated in a negative feedback regulatory manner during treatment of cells with arsenic trioxide, and that molecular or pharmacological inhibition of their activation enhances arsenic trioxide-dependent apoptosis and antileukemic responses (J Biol Chem. 283:12034–42, 2008, and Cancer Res. 66:6763–71, 2006). We examined the activation status of Mnk kinases in response to treatment of AML cells with chemotherapy and the function of these kinases in the generation of antileukemic responses. The human AML cell lines U937, K562, or MM6 were treated with cytarabine, in the presence or absence of a commercially available pharmacological Mnk-1 inhibitor (Calbiochem); and the phosphorylation of Mnk and its downstream effector, eIF4E, were assessed. Treatment with cytarabine increased phosphorylation of Mnk and eIF4E. The cytarabine-dependent eIF4E phosphorylation was blocked when human leukemia cell lines were treated with the pharmacologic Mnk inhibitor, indicating that Mnk regulates eIF4E activity. Such phosphorylation was also found to be defective in Mnk1/Mnk2 double knockout mouse embryonic fibroblast (MEF) cells, as compared to wild-type MEFs. Importantly, cytarabine-induced apoptosis was strongly enhanced in Mnk1−/− Mnk2−/− MEFs, as compared to Mnk1+/+Mnk2+/+ MEFs. To define the role of Mnk kinases in the generation of chemotherapy-induced antileukemic responses, human leukemia cell lines and bone marrow or peripheral blood mononuclear cells from patients with AML were used in clonogenic assays in methylcelluose to determine the effects of Mnk inhibition in the cytarabine-mediated leukemic progenitor (CFU-L) growth. The Mnk inhibitor potentiated the inhibitory effects of cytarabine on U937-derived CFU-L colonies and bone marrow or peripheral blood-derived CFU-L from 3 patients with AML. Interestingly, combinations of the Mnk inhibitor with the mTOR inhibitor, rapamycin, also resulted in more pronounced inhibitory effects on CFU-L colony formation than each agent alone. Altogether, these findings demonstrate that the Mnk pathway is activated during treatment of AML cells with cytarabine and that such activation occurs in a negative feedback regulatory manner to counteract the antileukemic effects of cytarabine. They also raise the possibility that targeting Mnk kinases may provide a novel approach to enhance the effects of chemotherapy on AML cells in vitro and possibly in vivo.
Treatment with Hsp90 Inhibitor, 17-AAG Overcomes Resistance to Small Molecule FLT3-Inhibitors in FLT3/ITD-Positive Leukemia Cells Harboring N676K-Mutation.
Authors: Minami Yosuke , Nomura Yuka , Abe Akihiro , Tanizaki Ryohei , Kuwatsuka Yachiyo , Kiyoi Hitoshi , Naoe Tomoki
Published: April 2020

The receptor tyrosine kinase FLT3 is mutated in ~30% of acute myeloid leukemia (AML) and small molecules that selectively inhibit FLT3 kinase activity induce apoptosis in blasts from AML patients with FLT3 mutations and prolong survival in animal models of FLT3-induced myeloproliferative disease. Therefore, targeting mutated FLT3 is an attractive therapeutic strategy, and early clinical trials testing FLT3-inihibitors have shown measurable clinical responses. However, most of these responses were transient and in some cases an additive single amino acid substitution (such as N676K) within FLT3 kinase domain was reported to be a possible cause for the temporary response. We previously showed a novel FLT3 inhibitor, FI-700, selectivity suppresses the growth of leukemia cells with FLT3 mutations (Kiyoi, et al., Clin Cancer Res 07). In order to examine how we can overcome drug resistance due to additional kinase mutations, we cultured MOLM-13 (FLT3/ITD-positive leukemia cell line) cells with increasing doses of FI-700 to generate several FI-700-resistant sublines. In 6 of 6 clones with resistance to more than 1 μM FI-700, FLT3N676K mutation was detected in one allele, and these were also resistant to small molecule FLT3-inhibitors such as AG1295 and AG1296. In a drug-screening for this kind of subline (MOLM-13/FLT3N676K), sub-molar treatment with molecular chaperon Hsp90 inhibitor, 17-AAG, inhibited growth as revealed by MTT-assays, and a combination of 17-AAG and FI-700 synergistically induced cell death as indicated by DNA-histograms and PI/Annexin-V assays. Expression of FLT3 by FACS and phosphorylation of FLT3 and its substrate molecules (such as STAT5), were significantly inhibited by the combined treatment of FI-700 and 17-AAG. To reveal the detailed mechanisms underlying these synergistic effects, we are currently investigating the binding of FLT3 and Hsp90, the degradation and the localization of FLT3 in these cells. We are also investigating the in vivo-effects of combined treatment in NOD/SCID mice inoculated with MOLM-13/FLT3N676K. These results imply that treatment of FLT3/ITD-positive leukemia cells with 17-AAG is a promising strategy for overcoming the drug resistance of cells with mutations in the FLT3 kinase domain.
GF-15, a Novel Inhibitor of Centrosomal Clustering, Suppresses Tumor Growth in Vivo.
Authors: Raab Marc S , Breitkreutz Iris , Rebacz Blanca , Nguyen Thu , Hayden Patrick J. , Clausen Mads H , Larsen Thomas O. , Fruehauf Johannes , Anderson Kenneth C. , Kraemer Alwin
Published: April 2020

The lack of tumor-specific targets that allow for selective eradication of malignant cells without affecting healthy tissues is a major drawback of cancer chemotherapy. In contrast to normal cells, tumor cells frequently contain multiple centrosomes, associated with the formation of multipolar mitotic spindles and chromosome segregation defects. In many tumor types, mitotic stability is regained after clonal selection by coalescence of multiple centrosomes into two functional spindle poles (centrosomal clustering). To identify potent and selective inhibitors of centrosomal clustering, we recently described a phenotype-based small molecule screening strategy directed to induce tumor cells with supernumerary centrosomes to undergo multipolar mitoses, thereby resulting in apoptotic cell death (Rebacz et al., Cancer Res2007; 67: 6342–6350). We here describe the novel small molecule GF-15, which is a derivative of griseofulvin and a potent inhibitor of centrosomal clustering, thereby inducing multipolar spindles followed by apoptosis. We tested more than 25 cancer cell lines from hematologic malignancies (including acute and chronic leukemias, lymphomas and multiple myeloma) as well as solid tumors (including glioblastoma, colon, cervix, and pancreatic cancers) and found mean inhibitory concentrations (IC50) for proliferation and survival in the range of 1–5μM GF-15, associated with activation of caspases 8, 9, and 3. As expected, tumor cell lines displaying only limited centrosomal aberrations or microsatellite instability were less sensitive to treatment with GF-15. Importantly, non-malignant cells without supernumerary centrosomes including PBMCs, immortalized hepatocytes, and bone marrow stromal cells, did not reach their IC50 even at 30μM GF-15. Specifically, cell cycle analysis of synchronized tumor cells showed marked G2/M arrest, followed by a dramatic increase of the sub-G1 fraction, after treatment with GF-15. In addition, short term treatment with GF-15 was also associated with inhibition of cytokine-triggered cell migration. Finally, both intraperitoneal and oral GF-15 treatment of murine xenograft models of human colon cancer and multiple myeloma resulted in tumor growth inhibition and significantly prolonged survival in vivo. Taken together, our results demonstrate the in vitro and in vivo anti-tumor efficacy of a first in class small molecule inhibitor of centrosomal clustering with specificity for tumor cells, and strongly support its clinical evaluation to improve patient outcome in both hematologic malignancies and solid tumors.