bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular Characterization Of Adult T-Cell Leukemia/Lymphoma
Authors: Nagata Yasunobu , Kitanaka Akira , Sanada Masashi , Sato Aiko , Okuno Yusuke , Shiraishi Yuichi , Chiba Kenichi , Tanaka Hiroko , Yoshizato Tetsuichi , Kon Ayana , Yoshida Kenichi , Ishiyama Ken , Miyawaki Shuichi , Miyano Satoru , Shimoda Kazuya , Ogawa Seishi
Published: October 2019

Adult T-cell leukemia/lymphoma (ATL) is an aggressive form of peripheral T-cell lymphoma (PTCL), which is etiologically associated with human T-lymphotropic virus type I (HTLV-1) infection during early infancy. Although HTLV-1 can effectively immortalize T-cells, there is a long latency period of ∼50 years prior to the onset of ATL, suggesting that HTLV-1 infection alone may not be sufficient for the development of ATL, but additional acquired genetic hits that occur in immortalized T-cells during the later life are essential for its pathogenesis. However, little has been known about those genetic hits that are involved in the pathogenesis of ATL.

The purpose of this study is to understand the genetic basis of ATL, 32 cases with different ATL subtypes, including acute (N=15), chronic (N=6), lymphoma (N=10), smoldering (N=1) types were analyzed by whole exome sequencing as well as copy number analysis. With a mean coverage of 119, 94% of the target sequences were analyzed at more than 20 depth on average. A total of 2,862 somatic changes were detected in 32 cases with a true positive rate of 99% (329 of the 334 tested were confirmed by PCR-based deep sequencing). These consisted of 2,512 missense mutations, 174 nonsense mutations, 65 splice-site mutations, and 111 indels. The mutation rate of 89 (44-227) per sample was significantly higher than that in acute myeloid leukemia (7.3–13), myelodysplastic syndromes (9.2) and chronic lymphocytic leukemia (11.5). Recurrent mutations were observed in 350 genes, of which 192 were considered to be significantly mutated (q < 0.05) compared to background mutation rates (1.79 mutations per megabase).

To investigate significantly mutated pathways, each pathway registered in the Kyoto Encyclopedia of Genes and Genomes, BioCarta, Reactome, Sigma-Aldrich and Signaling Transduction KE was tested on the basis of the background mutation rate observed in whole-exome sequencing data, which revealed a number of significantly mutated functional pathways, including pathways involeved in T cell receptor signaling, leukocyte trans-endothelial migration, VEGF and WNT signalings and other signaling pathways.

Genes for epigenetic regulations were also among the frequent targets of gene mutations. We performed targeted deep sequencing of TET2, IDH1/2 and DNMT3A in 182 ATL cases. In total, 19 TET2 mutations were identified in 16 cases (8.7%). Different subtypes of ATL were almost evenly affected with 9 out of 67 acute, 3 out of 42 chronic and 4 out of 56 lymphoma types having TET2 mutations. Less frequent mutations of IDH2 and DNMT3A (both 1%) were also identified. Our findings on genetic alterations provide a novel insight into the pathogenesis of ATL.

Disclosures:

No relevant conflicts of interest to declare.
Interrogation Of Signaling Networks In B Cell Malignancies To Improve Classification Of Patient Subsets For Targeted Therapy
Authors: Obidi Joyce , Burke Patricia , Goswami Trishna , Richman Laura , Mendel Dirk , Bao Haifeng
Published: October 2019

Targeted therapies have demonstrated promising advances in treatment of B cell malignancies. The premise of this project is that patients with closely related signaling “fingerprints” will have similar biological and clinical features. The objectives were to identify signaling profiles that are associated with clinical features and classify patients based on a signaling signature. 19 DLBCL and CLL cell lines with different molecular features were assessed in this study to establish relevant signaling features. We examined chronic BCR signaling, which leads to the activation of several downstream signaling pathways such as MAPK, AKT and NFκB pathways, in DLBCL and CLL cell lines. Phosphoflow cytometry was used to profile cell lines; a panel of 2 stimulation conditions was combined with 3 phospho-protein readouts. Basal level signal activities in cells were compared to enhance activation with PMA/IONO and F(ab’)2 IgG/IgM. We observed that AKT signaling in cell lines contrasted significantly with signaling in Normal B cells (CD20+ PBMCs). Constitutive activation of the AKT signaling pathways at baseline was observed in DLBCL cell lines; very high basal phosphorylated Ribosomal Protein S6 was observed in ABC (activated B-cell) phenotype DLBCL cell lines. In addition, upon ex vivo stimulation 30% of the cell lines demonstrated high level of pathway activity when compared to normal B cells. In this study we have identified 2 distinct signaling profiles based on BCR signaling in DLBCL cell lines; 7 of 12 DLBCL cell lines demonstrated impaired BCR signaling and 4 of 12 cell lines were hyper responsive to BCR stimulation. In the case of NFκB signaling, ex vivo stimulation revealed an absence of functional NFκB activity in the several cell lines. However, the lack of NFκB pathway activity observed in these cell lines was restored with the use of a phosphatase inhibitor, indicating that impaired signaling is not through loss of kinase function but is due to an increase in negative regulation. We also explored inhibition of pathway activities in response to small molecule inhibitors targeting BCR signaling. The inconsistency in MAPK, AKT, and NFκB pathway activities observed could aid in defining a signaling profile applicable to patient stratification. Overall, our results suggest that profiling of signaling network activities represents a promising approach to molecularly classifying DLBCL and CLL subtypes.

Disclosures:

Obidi: MedImmune: Employment. Burke:MedImmune: Employment. Goswami:MediImmune: Employment. Richman:MedImmune: Employment. Mendel:Medimmune: Employment. Bao:MediImmune: Employment.
25-OH-Vitamin-D Deficiency Impairs Rituximab-Mediated Cellular Cytotoxicity and Is Associated With An Inferior Outcome Of Elderly DLBCL Patients Treated With Rituximab
Authors: Bittenbring Joerg Thomas , Altmann Bettina , Neumann Frank , Achenbach Marina , Reichrath Joerg , Ziepert Marita , Geisel Juergen , Regitz Evi , Held Gerhard , Pfreundschuh Michael
Published: October 2019

Background

To investigate the impact and underlying mechanisms of vitamin-D-deficiency (VDD) on outcome of elderly (61 to 80 year-old) DLBCL patients.

Methods

Pretreatment 25-OH-vitamin-D serum levels from 359 patients treated in the prospective multicenter RICOVER-60 trial with 6 or 8 cycles of CHOP-14 with and without 8 cycles rituximab and 63 patients in the RICOVER-noRT study treated with 6xCHOP-14 + 8xR were determined determined by LIASION®, a commercially available chemoluminescent immunoassay.

Results

RICOVER-60 patients with VDD (defined as serum levels ≤8 ng/m l) and treated with rituximab had a 3-year event-free survival of 59% compared to 79% in patients with >8 ng/ml; 3-year overall survival was 70% and 82%, respectively. These differences were significant in a multivariable analysis adjusting for IPI risk factors with a hazard ratio of 2.1 [p=0.008] for event-free survival and 1.9 [p=0.040] for overall survival. In patients treated without rituximab 3-year EFS was not significantly different in patients with vitamin-D levels ≤8 and >8 ng/ml (HR 1.2; p=0.388). These results were confirmed in an independent validation set of 63 patients treated within the RICOVER-noRT study. Rituximab-mediated cellular toxicity (RMCC) against the CD20+ cell line Daudi as determined by LDH release assay increased significantly (p<0.005) in 5/5 vitamin-D-deficient individuals after vitamin-D substitution and normalization of their vitamin-D levels.

Conclusions

VDD is a significant risk factor for elderly DLBCL patients treated with rituximab. Our results show that VDD impairs RMCC and that RMCC can be improved by vitamin-D substitution. This together with the differential effect of VDD in patients treated with and without rituximab suggests that vitamin-D substitution might result in a better outcome of these patients when treated with CHOP plus rituximab. Supported by a grant from Deutsche Krebshilfe.

Disclosures:

No relevant conflicts of interest to declare.
Consumption Of FVIII Concentrate During On-Demand and Prophylactic Treatment With Human-cl rhFVIII In Prospective Clinical Studies In Adult Patients With Severe Hemophilia A
Authors: Tiede Andreas , Knaub Sigurd , Oldenburg Johannes , Bichler Johann
Published: October 2019

Background

There is ample evidence to support prophylactic treatment with factor VIII (FVIII) in children with severe hemophilia A (HA). Adults with severe HA are often treated on-demand and the potential benefit of regular prophylaxis is linked to a higher consumption of FVIII concentrates. During the clinical development of Human-cl rhFVIII, the first recombinant FVIII concentrate from a human cell line, its efficacy and safety was evaluated in previously treated adult patients (PTPs) during on-demand treatment only (GENA-01) and prophylaxis (GENA-08).

Aims

To compare post-hoc the annualized bleeding rate (ABR) and the consumption of FVIII concentrate in patients treated exclusively on-demand with those treated prophylactically.

Methods

Both prospective multi-centre studies were approved by the Ethics Committees of each participating institution and informed consent was obtained from the patient prior to any trial activity. In GENA-01, patients were to be treated on-demand for ≥6 months and ≥50 exposure days with protocol recommended doses ranging from 20 to 60 IU/kg, depending on the severity of the bleed. In GENA-08, patients were to be treated prophylactically with Human-cl rhFVIII every other day with 30-40 IU/kg for ≥6 months. Human-cl rhFVIII was also to be used in case of breakthrough bleeds.

Results

22 PTPs with severe HA were enrolled in GENA-01, and 32 in GENA-08. The study populations were reasonably well comparable to each other (GENA-01 vs. GENA-08, mean±SD), regarding age (39.6±14.1 vs. 37.3±13.6 years), body mass index (23.9±4.8 vs. 25.8±4.9 kg/m2), hemophilia joint health score (38.4±30.3 vs. 34.6±32.2), race (>80% White in both studies) and historical bleeding sites. In GENA-08, the majority of patients (65.6%) had been treated prophylactically prior to study entry. Their historical mean±SD ABR was 6.6 ±11.3 (median: 2.0, range: 0-48.7) and their mean prophylactic dose/month was 293 IU/kg. The other 11 patients who had been treated on-demand had a mean±SD ABR of 47.4±34.6 (median: 36.5, range: 12.2-121.7). In GENA-01, all but 2 patients were treated on-demand prior to study entry. The historical mean±SD ABR of all GENA-01 patients was 49.5±35.9 (median: 44.6, range: 2.0-158.7). The ABR and FVIII consumption during the studies are shown in Table 1.

Conclusion

The data suggest that regular prophylactic treatment with Human-cl rhFVIII in adult PTPs with severe HA resulted in an approximately 25-fold reduction of bleeding rate, and a 3-fold increase of FVIII concentrate consumption.

Disclosures:

Tiede: Octapharma AG: Consultancy, Investigator Other. Knaub:Octapharma AG: Employment. Oldenburg:Octapharma AG: Consultancy, Investigator Other. Bichler:Octapharma AG: Employment.
Latest Results From The PUP-GCP Clinical Trial: A Low Inhibitor Rate In Previously Untreated Patients With Severe Hemophilia A Treated With Octanate
Authors: Klukowska Anna , Jansen Martina , Komrska Vladimir , Laguna Pawel , Vdovin Vladimir , Knaub Sigurd
Published: October 2019

Background

Octanate is a highly purified, double virus inactivated, human plasma-derived factor VIII (FVIII) concentrate with all coagulation FVIII bound to its natural stabilizer VWF in a VWF:RCo/FVIII:C ratio of approximately 0.4. Five prospective GCP studies with octanate were conducted in 77 previously treated patients (PTPs) with severe hemophilia A. None of these 77 PTPs developed an inhibitor while treated exclusively with octanate.

Aim

To assess the immunogenicity in previously untreated patients (PUPs), a prospective clinical trial has been initiated in 2000. This included 50 PUPs with severe hemophilia A for an observational period of 100 exposure days with octanate, for at least 6 months.

Methods

Patients with severe hemophilia A without previous exposure to FVIII or FVIII-containing products were enrolled. Efficacy and tolerability were assessed by a 4-point verbal rating scale. Inhibitors were assessed according to modified Bethesda method prior to treatment every 3-4 exposure days (ED 1-20), and after treatment every 10 EDs (ED 21-100), but at minimum every three months.

Results

Two of 50 (4%) subjects developed clinically relevant inhibitor titers over the course of the study. Another two displayed inhibitors that disappeared spontaneously without change of dose or dosing interval. All inhibitors developed under on-demand treatment and before ED 50. From the 50 subjects, 42 had exceeded 50 EDs at the time of this analysis. Octanate was well-tolerated and the adverse event profile was consistent with the population studied. The hemostatic efficacy in prophylaxis and treatment of bleeding episodes was generally rated as “excellent” and no complication was reported for any surgical treatment.

Conclusion

Despite frequent inhibitor testing and predominant on-demand treatment, the data indicate a low overall inhibitor rate for octanate in patients who exceeded 50 exposure days (4/42) of which only 2 (4.8%) were clinically relevant.

Disclosures:

Klukowska: Octapharma AG: Investigator Other. Jansen:Octapharma AG: Employment. Komrska:Octapharma AG: Investigator Other. Laguna:Octapharma AG: Investigator Other. Vdovin:Octapharma AG: Investigator Other. Knaub:Octapharma AG: Employment.
The Leukemia-Associated Rho Guanine Nucleotide Exchange Factor (LARG) /ARHGEF12 Is Important For Erythroid Differentiation
Authors: Gao Li , Zhou Yong , Tang JingYan , Shen Shuhong
Published: October 2019

ARHGEF12,which was initially identified as a fusion partner of MLL in acute myeloid leukemia, is a regulatory protein involved in the GDP/GTP exchange reaction of the Rho A and activates a Rho-GTPase-dependent signaling pathway. However, the role of ARHGEF12 in hematopoiesis remains unknown. Here, we first report that ARHGEF12 takes part in erythroid differentiation not only in erythroleukemia cell line K562 but also in zebra fish based on knockdown technology. Although primitive hematopoiesis including erythroid differentiation progressed normally, double silencing of both orthologues arhgef12a and arhgef12b in Danio rerio with morpholino caused erythropenia during definitive hematopoiesis. Meanwhile, cytology assay reveal less mature red blood cells with large and loose nuclear in morpholino injected embryos. The phenotype of blocked erythroid differentiation can be rescured by mRNA overexpression of RhoA active mutant. On the other hand, injection of mRNA transcripted from the dominant negative mutant of RhoA could replicate the phenotype of arhgef12 silencing. Antibody array screen of kinase phosphorylation suggested STAT1 and MAPK pathways downstream of Rho A may be important for erythroid differentiation in our modles.

Disclosures:

No relevant conflicts of interest to declare.
Human NUP98/Iqcg Fusion Gene Induced An Acute Myelomonocytic Leukemia In Mice
Authors: Zhang Qi-Yao , Liu Ping , Pan Mengmeng , Chen Sai-Juan
Published: October 2019

NUP98 gene encoding a nucleoporin located at 11p15 has been reported to be fused with about 30 different partner genes by chromosomal translocations in hematological malignances. NUP98/IQCG was cloned in a patient of Myeloid/ T-lymphoid bi-phenotypic leukemia with 47, XX, t(3;11)(q29q13;p15)der(3)(q29), +21 karyotype. However, NUP98/IQCG’s ability for leukemogenesis has not been identified yet.In this study, we established a retrovirus-mediated murine bone marrow transduction and transplantation (BMT) model of NUP98/IQCG to investigate its oncogenicity. In our model, half of the NUP98/IQCG mice developed a penetrable and transplantable acute myelomonocytic leukemia ,which was similar to the phenotype of patient with t(3;11). It suggested that NUP98/IQCG could induce the disease development.

To investigate how the fusion gene promoted leukemogenesis, we transduced NUP98/IQCG into primary bone marrow cells, and found that NUP98/IQCG-expressing cells retained the ability to generate colonies in serial replating, while seldom control cells did, which indicated the increasing self-renewal ability caused by NUP98/IQCG. Meanwhile, when induced by Macrophage-Colony Stimulating Factor, NUP98/IQCG -expression bone marrow cells showed enhanced proliferation in vitro. Further molecular mechanism studies revealed that NUP98/IQCG could be involved in both NFκB and CREB pathways during leukemia development.

In summary, we showed that NUP98/IQCG promoted leukemogenesis in BMT mouse model through increasing the bone marrow cells’ proliferation and self-renewal capacity, which explained the fusion gene’s oncogenicity in patient with t(3;11). Our mouse model will be a powerful tool both to investigate the leukemogenic mechanism of NUP98-related fusion gene, and to find the drugs for treating the disease.

Disclosures:

No relevant conflicts of interest to declare.
Ezh2 Is An Essential Regulator Of T-Cell Development and Oncogenic Transformation
Authors: Simon Camille , Chagraoui Jalila , Krosl Jana , Hebert Josee , Sauvageau Guy
Published: October 2019

Enhancer of zeste homolog 2 (EZH2) catalyzes di- and trimethylation of lysine 27 on histone H3 (H3K27me2/3) and establishes chromatin marks associated with gene silencing. We and others have recently shown that Ezh2 and its partners act as tumour suppressor genes in mouse and likely human lymphoblastic leukemia. Moreover some studies also suggest that Ezh2 is strongly required during B and T cell differentiation. However, the function of EZH2 during these processes remains unclear. For functional study we exploited an Ezh2 conditional knockout mouse model. The Cre-mediated deletion generates a mutated Ezh2Δ allele and abrogates production of EZH2 protein. Upon gene inactivation we monitored T-cell maturation and cancer development. We found that Ezh2 inactivation induces a block at the DN3-DN4 transition of TCRab+T-cells while TCRγδ T-cells were increased by 5 fold compared to wild type animals. Cell cycle analysis revealed increase in the proportions of TCRγδ+T-cells in the G2 phase compared to TCRβ+T-cells and wild type controls. This observation suggested a possibility of G2/M checkpoint activation resulting from either improper DNA replication, or a non-repaired DNA damage. Moreover we found that the Ezh2 deficient TCRγδ+ leukemia were prone to genomic instability. A majority of leukemias analyzed were aneuploid, and ∼50% were near-tetraploid. These observations were confirmed by Spectral Karyotyping (SKY), which also enabled detection of several chromosomal rearrangements. Consistent with these observations, analysis of global gene expression data from various RNA-Seq-derived datasets revealed that the genes having the highest correlation factor with Ezh2 are involved in cell division, DNA replication and DNA damage repair. Together, these studies show that Ezh2 is an essential regulator of the TCRγδ T-cell state, and prevents T-cell transformation, likely through regulation of DNA replication, cell division or DNA damage repair.

Disclosures:

No relevant conflicts of interest to declare.
ABR, a Novel Inducer Of Transcription Factor C/EBPα, Is Necessary For Myeloid Differentiation and a Prognostic Factor In Acute Myeloid Leukemia
Authors: Namasu Carolina Yaeko , Gerloff Dennis , Wurm Alexander Arthur , Braeuer-Hartmann Daniela , Hartmann Jens-Uwe , Katzerke Christiane , Schwind Sebastian , Hilger Nadja , Fricke Stephan , Niederwieser Dietger , Behre Gerhard
Published: October 2019

ABR (Active BCR-related) is the only protein in humans and mice closely homologous to BCR. BCR acts as a tumor suppressor in different cancers, such as chronic myeloid leukemia and meningiomas. A putative anti-oncogenic role of ABR has been shown in tumors of the central nervous system, such as medulloblastoma and astrocytomas, in which deletion of ABR was found. However, the role of ABR in hematopoiesis or leukemia remains unclear. We hypothesized that ABR might be important for myelopoiesis via increasing the expression of C/EBPα, a transcription factor known to be pivotal for myeloid differentiation and functionally impaired in acute myeloid leukemia (AML). In fact, we found that ABR expression is dramatically down-regulated (Figure 1, p=0.01) in bone marrow from AML patients (pts; n=63) compared to bone marrow (BM) mononuclear cells from healthy donors (n=3). In agreement with this finding, Abr is significantly increased during M-CSF and G-CSF-stimulated differentiation of primary wild type mouse BM cells (p<0.05). Additionally, we observe that ABR is necessary for monopoiesis induced by PMA (phorbol 12-myristate 13-acetate), since ABR knockdown in leukemic U937 cells results in a significant reduction of about 50% in the number of CD11b+ cells 48h after PMA treatment (p<0.05). Enforced ABR expression induces C/EBPα and its targets M-CSFR, G-CSFR and microRNA (miR)-223 in U937 cells (p<0.01). Moreover, we prove that ABR knockdown prevents induction of CEBPA, M-CSFR and G-CSFR during PMA-mediated differentiation (p<0.05). ABR overexpression blocks cell-cycle progression and down-regulates the known C/EBPα inhibitor E2F1 (p<0.01) in U937 cells, indicating the functional role of ABR as tumor suppressor. Those data suggest that ABR might induce CEBPA expression via inhibition of cell cycle activator E2F1. Finally, we are the first to identify ABR as a good prognostic factor in AML: patients with high ABR expression (median cut) survive significantly longer after allogeneic hematopoietic stem cell transplantation (Figure 2, p=0.04, log-rank test). Furthermore, high ABR expression associates with a low percentage of blasts in the peripheral blood (p=0.006) and high levels of antileukemic miR-181a (p<0.001). In conclusion, these data indicate that ABR, a novel inducer of C/EBPα, is necessary for myelopoiesis and a prognostic factor in AML. Raising ABR levels might be a goal for future therapeutics in AML.

Disclosures:

No relevant conflicts of interest to declare.
Induced Dependence On The ALK Kinase Inhibitor Crizotinib In Formerly Sensitive Anaplastic Large Cell Lymphoma Cells
Authors: Amit Amin Dipak , Pongtornpipat Praechompoo , Schatz Jonathan H.
Published: October 2019

Kinase inhibitors can be highly effective against cancers driven by specific oncogenic kinase proteins, but resistance usually develops after prolonged treatment. Target-dependent mechanisms, typically acquired second-site mutations that prevent drug binding, or target-independent mechanisms, such as downstream or parallel pathway activation, may mediate resistance. Prolonged exposure of kinase-dependent cell lines to inhibitors in vitro to select for resistant populations has identified clinically relevant resistance mechanisms against a number of inhibitors. ALK-positive anaplastic large-cell lymphoma (ALCL) is an uncommon form of peripheral T-cell lymphoma (PTCL) driven by the NPM-ALK fusion kinase, resulting from a t(2;5)(p23;q35) chromosomal rearrangement. ALK inhibitors developed for lung cancer, where a different ALK fusion (EML4-ALK) is found in 6% of cases, have shown promise clinically as a new treatment modality for ALK+ ALCL patients. In this study, we selected three NPM-ALK-dependent ALCL cell lines (Karpas-299, SUP-M2, and SU-DHL-1) for resistance to the ALK inhibitor crizotinib. All three lines initially were highly sensitive the drug (IC50< 100 nM) and required months of exposure at gradually increasing concentrations before acquiring resistance. Previous work has identified specific ALK kinase second-site mutations that promote crizotinib resistance, which we also observed in some resistant lines generated. A unique situation arose, however, in a sub-clone of Karpas-299, that was able to grow in crizotinib at concentrations > 1 µM (Karpas-299CR1000). This line had greatly increased viability in crizotinib at concentrations up to 1 µM, compared to untreated cells, suggesting the drug actually maintained growth. Plated in 250 nM crizotinib, Karpas-299CR1000 grows rapidly, similar to the parent line growing in drug-free media. Plated in drug-free media, however, the line’s viability drops rapidly to zero and dies, similar to the parent line plated in 250 nM crizotinib. Therefore the drug that once shut off the signaling necessary for the cells’ survival, after prolonged exposure (>9 months), became necessary to maintain it in the sub-clonal population. Melanoma cells driven by BRAF-V600E selected for vemurafenib resistance had a similar phenotype in a recent report (Das Thakur, et al., 2013). These cells amplified mutant BRAF to overcome the inhibitor but came to require the drug to counteract BRAF over-activity with amplification beyond a certain point. Preliminarily, we find a similar effect of NPM-ALK amplification in Karpas-299CR1000 cells but are actively exploring mechanism. In sum, our study shows that long-term exposure of ALK+ ALCL cells to an ALK inhibitor may induce a resistant phenotype that comes to depend on drug presence to prevent over-activity of the mutant kinase.

Disclosures:

No relevant conflicts of interest to declare.
Activities Of Commonly Used Tyrosine Kinase Inhibitors Against Wild-Type C-KIT and Potential Impact On Normal Hematopoiesis
Authors: Galanis Allison , Levis Mark
Published: October 2019

The receptor tyrosine kinase (RTK) c-KIT is important in early hematopoiesis, and impaired c-KIT activity is associated with abnormal erythroid and megakaryotic function in animal models. Several approved and investigational small molecule tyrosine kinase inhibitors have off-target activity against c-KIT, with resultant potential for hematopoietic toxicity. To investigate the association between c-KIT inhibition by TKIs and myelosuppression, we determined the inhibitory activity of a series of TKIs against c-KIT using immunoblot estimates of IC50 concentrations, and compared those results with the compounds’ effects on human hematopoiesis in progenitor cell assays. For this study, we used quizartinib, crenolanib, sorafenib, imatinib, pazopanib, and dasatinib. IC50s against FLT3 and c-Kit were established for each kinase with assays performed both in culture medium (RPMI/10%fetal bovine serum) and 100% human plasma. Colony formation assays with normal human bone marrow were performed in quadruplicate with clinically achievable levels of each drug, adjusting for plasma protein binding (and based on published pharmacokinetic data from plasma samples of human subjects). Out of the six TKIs studied, most showed limited to no inhibition of the formation of colony-forming unit-granulocyte macrophage (CFU-GM). Three TKIs, quizartinib, pazopanib, and dasatinib, demonstrated inhibition of burst-forming unit-erythroid (BFU-E) colonies. The highest doses of these drugs inhibited growth of BFU-E colonies by 30% versus untreated control. These same three drugs are also the most potent c-Kit inhibitors of this group, with IC50s of 35, 7.5, and 1.4 nM, respectively, in culture medium. The remaining TKIs, crenolanib, sorafenib, and imatinib, showed far less BFU-E inhibition, none inhibiting colony formation by more than 25%. These TKIs also had higher c-Kit IC50 values of 65, 395, and 122 nM, respectively. We conclude that c-KIT inhibition correlates with eythroid progenitor cell suppression in vitro, and that TKIs that achieve c-KIT inhibitory levels in vivo would be predicted to cause anemia. During clinical development of TKIs that have c-KIT inhibitory activity, attention should be paid to the therapeutic index between the targeted kinase (e.g., FLT3) and c-KIT, so as to avoid unnecessary myelosuppresive effects. Dose range determinations to spare c-Kit inhibition would lead to a decreased risk of disrupting normal hematopoiesis, making treatment far more manageable for these patients.

Disclosures:

Levis: Ambit Biosciences: Consultancy.
Oxidation Of Peroxiredoxins By Thioredoxin Inactivation Is Mediated In Arsenic Trioxide-Induced Reactive Oxygen Species Formation and Its Cytotoxicity In Acute Promyelocytic Leukemia Cells
Authors: Mun Yeung-Chul , Ahn Jee-Young , Yoo Eun-Sun , Huh Jungwon , Lee Kyoung Eun , Nam Eunmi , Lee Soon Nam , Seong Chu-Myong
Published: October 2019

Backgrounds

The Arsenic trioxide (ATO) is an effective cancer therapeutic drug for acute promyelocytic leukemia (APL). ATO exerts its effect mainly raising oxidative stress. However, not only the mechanisms of reactive oxygen species (ROS) generation by ATO but involvement of redox enzymes including peroxiredoxin (PRX) and thioredoxin (TRX) remains elusive. Aim of current study is to elucidate the mechanism of redox enzymes to elevate ROS during ATO-induced apoptosis in APL-derived NB4 cells.

Methods

NB4 cell line, which is one of the human acute promyelocytic leukemia cell lines, was cultured in RPMI-1640 medium supplemented with 10% FBS in CO2 humidified atmosphere at 37°C. NB4 cells were cultured with 2 μM arsenic trioxide to induce apoptosis for 16-48 hours. Apoptosis was measured by staining with 7-amino-actinomycin D (7-AAD) with flow cytometry. 2, 7-dichlrodihydro-fluorescein-diacetate (H2DCF-DA) and MitoSOX Red were used to detect cellular and mitochondrial ROS. SO2 form for PRX I, PRX II, and PRX III was detected by western blot assay using PRX SO2 form-specific antibody. Monomer/Dimer assay for PRX I, PRX II, PRX III, and TRX I was performed by western blot using non-reducing gel.

Results

Intracellular ROS of NB4 cells was increased significantly after 16 hour of ATO but decreased after 24 hour of ATO. Mitochondrial ROS of NB4 cells was increased significantly after 39 hour of ATO. Apoptosis of NB4 cell after ATO treatment was increased as time elapsed (24% on 16hr, 26% on 24hr, 48% on 39hr, and 60% on 48hr). Monomer, indicated active and reduced form, of peroxiredoxins was decreased and cysteine sulfinic acid (CP–SO2H) peroxiredoxins, indicated inactive and oxidized peroxiredoxins, was increased in NB4 cells after ATO treatment as time goes by. Similarily, monomer of thioredoxin-1 (active thioredoxin) was decreased and multimer of thioredoxin-1 (inactive thioredoxin) was increased in NB4 cells after ATO treatment as time elapsed.

Conclusions

Our data showed inactivation of peroxiredoxins by oxidation was developed during ATO-induced ROS generation and APL cell apoptosis. These peroxiredoxins oxidation was probably due to increment of reduced thioredoxin in NB4 cells after ATO treatment. These findings suggest ATO-induced anti-leukemic activity is more likely due to a TRX system-mediated cellular redox changes. Our study may provide the insights for finding novel targets in the development of new therapies, which potentiate ATO-induced apoptosis in APL cells.

Disclosures:

No relevant conflicts of interest to declare.
A Prospective, Multicenter Phase II Study Of Continuous Infusion Of FLAG For Patients Older Than 60 Years With Resistant Acute Myeloid Leukemia: A Comparison With Intensive Younger patients’ Trial
Authors: Kim Hawk , Lee Je-Hwan , Joo Young-Don , Bae Sung-Hwa , Lee Jung-Hee , Kim Dae-Young , Jo Jae-Cheol , Park Jae Hoo , Lee Won Sik , Ryoo Hun-Mo , Park Sang-Kyu , Lee Kyoo-Hyung
Published: October 2019

Relapsed/refractory acute myeloid leukemia (R/R AML) is hard to treat especially in elderly patients. We previously assessed continuous infusion (CI) of fludarabine and cytarabine plus idarubicin (CI-FLAG2) for patients under 65-years old with R/R AML. Also we started prospective phase II study of attenuated version of CI-FLAG2 for elderly patients (C-FLAG). R/R AML in elderly (¡Ã60 years old) patients were eligible. Induction chemotherapy consisted fludarabine and cytarabine (ARAC) as a 24-hr CI without idarubicin. Total 38 and 68 patients were enrolled in CI-FLAG1 and CI-FLAG2, respectively. There were no differences in terms of patients’ characteristics except for median age (p<0.001), HCT prior to salvage (p=0.002), WBC>20K/uL at salvage (p=0.004) and PB blast>40% at salvage, all which factors were unfavorable in C-FLAG. When comparing outcomes between CI-FLAG2 and C-FLAG, there were no difference in terms of CR rate (p=0.572) and objective response rate (ORR; p=0.899). Treatment failure patterns were also similar between C-FLAG and CI-FLAG2 (p=0.742). The most common treatment failure was resistant (66.7%) in C-FLAG. There were more frequent HCT in CI-FLAG2 (p<0.001) and consolidation chemotherapy in C-FALG (p=0.001). Poor predictors on ORR in C-FLAG were PB WBC>20K/uL at salvage (p=0.024) and PB blast >0% on early evaluation (p=0.013) by multivariate analysis. The overall survival of patients who achieve CR/CRp/CRi showed significantly prolonged survival compared with patients who did not in C-CLAG (p<0.001; Figure 1A). The median overall survivals were similar between CI-FLAG2 and C-FALG (p=0.427; Figure 1B).Figure 1Overall survivalFigure 1. Overall survival

Attenuated salvage regimen C-FALG in elderly patients was as effective as more intensive younger patients’ regimen CI-FALG2 in terms of response and survival although elderly patients had more unfavorable clinical characteristics.

Disclosures:

No relevant conflicts of interest to declare.
Analysis Of E2A Point Mutations In B Cell Leukemia
Authors: Soans Eroica , Choi John K
Published: October 2019

Introduction

TCF3 encodes for E2A protein, which belongs to the helix loop helix transcription factor family. E2A activates transcription of downstream genes by binding to E-box motifs as a homo or hetero dimer. E2A plays an important role in B lymphocyte development. Therefore deletion or mutations in TCF3 or even lowered activity of E2A are causes of B cell leukemia and lymphomas. Recently, three mutations V557E, D561E and N551K in E2A were isolated in Burkitt’s lymphoma (Schmitz, Young et al. 2012). The first two mutations are present in the homo dimerization region of E2A while N551K is present in the DNA binding region. Though the paper enumerated role of TCF3 in Burkitt’s lymphoma but the significance of these TCF3 mutations or mechanism needed further characterization. We hypothesized that these TCF3 mutations have an alternate mechanism as compared to wild type TCF3 and therefore may affect B cell development.

Methods

We characterized three TCF3 mutants by cloning them into in MIGR1 backbone using TOPO cloning. E2A activity was measured using an E2A-specific luciferase reporter assay in 293T cells. DNA binding activity was measured using a DNA protein binding colorimetric assay.

Results

V557E and D561E mutants have lower activity as compared to wild type E2A as studied using E2A-specific luciferase reporter assay; while N551K showed no activity in the same assay as compared to wild type E2A activity. Similarly V557E and D561 form weaker bonds with the E box motifs while N551K showed no DNA binding activity as studied using colorimetric DNA-protein binding assay. The plasmid expressions were verified using western blot analysis.

Conclusion

Our findings suggest mutations V557E and D561E may follow a similar pathway as wild-type E2A but have lower activity. The N551K mutation has an alternate pathway to wild type TCF3 that may impact B cell proliferation, survival and development.

Disclosures:

No relevant conflicts of interest to declare.
Cardiac Derived Stromal Cells Inhibit Tumor Cell Proliferation: A Potential Role Of miR206
Authors: McNiece Ian K. , Sivajothi Santhosh
Published: October 2019

Stromal cells play an important role in control of proliferation and differentiation of stem cells and are a key component of the stem cell niche. Bone marrow (BM) stromal cells (also termed mesenchymal stem cells; MSC) have been extensively studied and shown to control differentiation of hematopoietic stem cells (HSCs) in part through secreted growth factors. Recent studies have demonstrated the presence of stromal cells in cardiac tissue, however the role of cardiac stromal cells (CStrC) is unclear. In this study we have compared human CStrCs to human BM-MSCs and demonstrate that CStrCs have a similar morphology and surface marker expression as BM-MSCs. To further characterize the CStrCs we performed micro array analysis of human CStrCs compared to human BM-MSCs. The CStrCs expressed a distinct cytokine and cytokine receptor profile compared to BM-MSC. In addition, a number of micro RNAs were expressed at very high levels in CStrCs compared to BM-MSC. Cardiac- associated microRNAs, including miR-1, miR-133a, and miR-206 were expressed at higher levels in CStrCs compared to BM-MSC. Given the lack of tumor development in cardiac tissue we hypothesized that CStrCs would fail to support tumor cell growth which has been described for BM-MSCs. Therefore, we cultured human tumor cell lines on CStrCs and compared the tumor cell proliferation to BM-MSCs. CStrCs inhibited the proliferation of a range of tumor cell lines including myeloid cell lines HL60, K562, myeloma cell lines U-266, RPMI 8266, ARP-1 and the B cell line Raji, while BM-MSCs supported the proliferation of tumor cells. Further we tested media conditioned by CStrCs and demonstrated inhibition of proliferation of both cell lines. Previous studies have implicated miR-206 in inhibition of proliferation of tumor cells and given the high levels of expression of miR-206 in CStrCs we hypothesize that miR-206 is a key player in the inhibitory effects of CStrCs and this effect is mediated via secreted molecules.

Disclosures:

McNiece: Proteonomix Inc: Consultancy.
A Method To Identify Copy Number Aberrations (CNAs) From Whole Exome Sequence (WES) Data and Its Application To Multiple Myeloma Cell Lines and Patient Samples
Authors: Degenhardt Jeremiah D , Hoehn Kenneth B , Kwei Kevin A , Keats Jonathan J , Kirk Christopher J. , Tuch Brian B
Published: October 2019

Duplications and deletions of large-scale genomic regions, including whole chromosomes, are a hallmark of many malignant tumors. Gross cytogenetic changes of this type were among the earliest genomic lesions to be observed in tumors and represent an important predictor of outcome for many tumor types. In multiple myeloma, a core set of structural variants are regularly used for disease prognosis (e.g., del17p, t(4;14), t(14,16)). The presence of one or more of these lesions is predictive of a more aggressive disease type and poor outcomes for patients. The application of next-generation sequencing (NGS) to cancer samples has enhanced our ability to detect copy number aberrations (CNAs) and identify driver mutations. The NGS approach can generate higher resolution maps of these mutations than traditional methods, which in turn enables faster identification of the functionally relevant genes they harbor.

Most NGS-based methods for the identification of CNAs in tumors require whole genome sequencing (WGS) as input. Relatively fewer methods exist for the detection of CNAs from exome sequencing and, to our knowledge, these methods all rely on having matched normal data. However, WES is still far more common than WGS and matched normal samples are frequently not available. Therefore, we have developed a novel method for the identification of CNAs from single-sample WES data. Our method combines LOWESS smoothing & discrete-wavelet-transformation to normalize the exome coverage data with an HMM for coverage segmentation. We have applied our new method to WES data from 32 myeloma cell-lines for which aCGH copy number calls were also available for training and validation. Our method shows a high correlation with the aCGH calls (mean = 95%). We are currently applying the method to a set of 102 CD138+ tumor cell samples that were collected during patient screening on three Onyx-sponsored Phase 2 clinical trials of the proteasome inhibitor carfilzomib. We will report on the frequency of known and novel lesions in these samples, comparing to what we’ve observed in cell lines and other publically available CNA datasets.

Disclosures:

Degenhardt: Onyx Pharmaceuticals: Employment, Equity Ownership. Hoehn:Onyx Pharmaceuticals: Employment. Kwei:Onyx Pharmaceuticals: Employment, Equity Ownership. Kirk:Onyx Pharmaceuticals: Employment, Equity Ownership. Tuch:Onyx Pharmaceuticals: Employment, Equity Ownership.
A Novel Mutation In Bruton Tyrosine Kinase Confers Acquired Resistance To Ibrutinib (PCI-32765) In CLL
Authors: Furman Richard R. , Cheng Shuhua , Lu Pin , Setty Menu , Perez Alexandar , Guo Ailin , Racchumi Joelle , Ma Jiao , Coleman Morton , Venkataraman Kabaleeswaran , Xu Guozhou , Chen Wei , Wu Hao , Chang Betty Y. , Leslie Christina , Wang Y. Lynn
Published: October 2019

The Bruton tyrosine kinase (BTK) inhibitor, ibrutinib has produced durable remissions in chronic lymphocytic leukemia (CLL). We describe a CLL patient who progressed while receiving ibrutinib following 20 months of once daily dosing. A cysteine-to-serine amino acid replacement was identified in BTK at position 481 that disrupts the covalent, but not non-covalent, binding of ibrutinib to BTK in silico structural modeling1. The mutation was present in relapsed samples while absent in the pre-treatment and responding samples. Following the mutation, the B cell receptor (BCR) pathway was reactivated as evidenced by increased cell signaling activities and gene expression profiles. Comparing the relapsed samples with the pre-treatment and responding samples, at the cellular level, mutated CLL cells displayed higher levels of the cell proliferation marker Ki67 in vivo and higher levels of ex-vivo BrdU incorporation. Transfection of the C481S mutant construct into a sensitive lymphoma cell line rendered it much more resistant to ibrutinib treatment demonstrating the cellular impact of the mutation (see attached graph). Interestingly, the ibrutinib-resistant CLL cells remained sensitive to other BCR inhibitors such as dasatinib and SYK inhibitors. These results confirm BTK as an important pharmacologic target of ibrutinib. Further, a mechanism of resistance was revealed, and alternative therapeutic options for ibrutinib resistance were explored. (First three authors with equal contribution)

Disclosures:

Furman: Genentech: Consultancy, Speakers Bureau; GlaxoSmithKline: Consultancy, Speakers Bureau; Pharmacyclics: Consultancy; Gilead: Consultancy. Chang:Pharmacyclics: Employment, Equity Ownership.
Low-dose Triptolide could enhance the Idarubicin-induced apoptosis in KG1α cells through down-regulation of MDR1 and NF-κb
Authors: Xu Bing , Tang Jiahong , Chen Feili , Li Yanyan , Dong Huijuan , Liu Yuejian , Zhou Shuyun
Published: October 2019

Background

Triptolide (TPL), a diterpenoid triepoxide derived from Tripterygium wilfordii, has been used in Traditional Chinese Medicine (TCM) for centuries. Recently, TPL has also been shown to have strong anti-cancer ability both in vitro and in vivo. It could induce apoptosis in a variety of cancer cell lines. However, the clinical applications of TPL are limited by its narrow therapeutic window and severe toxicity. Thus, the focus of study about TPL has been switched to the ability of low dose of TPL in enhancing the drug-induced apoptosis of cancer cells. KG1a cell line is a kind of cell line which has been demonstrated to have a certain proportion of leukemia stem cells which are responsible for the relapse of leukemia. Usage of this kind of cell line could resemble the relapse situation of patients. MDR1 and NF-ΚB are two important factors closely related to drug-resistance. Dow-regulation of these two genes could lead to the sensitization of cells to drugs.

Aims

This study aims to explore whether low-dose TPL could enhance the Idarubicin-induced apoptosis in KG1a cells through down-regulation of MDR1 and NF-κB.

Methods

Hoechst 33342 staining and flow cytometry analysis were used to determine the ability of TPL in enhancing the Idarubicin-induced apoptosis in KG1a cells. RT-PCR as well as western blotting analysis were used to determine whether TPL combined with Idarubicin could down-regulate MDR1 and NF-κB.

Results

Hoechst 33342 staining and flow cytometry analysis showed that low-dose of TPL((IC20F5.0nM)) could significantly enhance the Idarubicin-induced apoptosis in KG1a cells(Idarubicin alone vs Idarubicn combined with TPL: 15.13%: 60.17%; P<0.05). TPL combined with Idarubicin could down-regulate expression of MDR1 both at protein and mRNA level with the down-regulation of NF-κB at protein level.

Conclusion

Low-dose Triptolide could enhance the Idarubicin-induced apoptosis IN KG1a cells through down-regulation of MDR1 and NF-κB

Disclosures:

No relevant conflicts of interest to declare.
Imatinib-induced apoptosis could be enhanced by Triptolide in Imatinib-resistant chronic myeloid leukemia cell line through down-regulation of HIF-1αand Nrf2
Authors: Xu Bing , Chen Feili , Liu Yuejian , Wang Shiyun , Li Rongwei , Dong Huijuan , Zha Jie
Published: October 2019

Background

The emergence of Imatinib has brought a new era for the treatment of chronic myeloid leukemia. However, resistant to Imatinib might lead to the treatment failure and death of patients. Thus, finding a drug which could enhance the Imatinib-induced apoptosis in vitro could provide the experimental base for the treatment of chronic myeloid leukemia. K562 cell line is a common cell line used in the study of chronic myeloid leukemia while K562/G cell line which is resistant to Imatinib is derived from K562 cell line.

Aim

This study aims to explore whether low-dose Triptolide(TPL) could enhance the Imatinib-induced apoptosis in K562/G cells and related mechanism.

Methods

K562/G cells were subjected to different treatments and thereafter MTT assay, flow cytometry and Western blot or RT-PCR were used to determine IC50, apoptotic status and expression of Nrf2, HIF-1α and their target genes.

Results

Triptolide is highly cytotoxic to K562/G cells in a concentration-dependent manner. To determine the combination effect of TPL and anticancer agents, K562/G cells were exposed to Imatinib(50μM) with or without TPL (25nM) for 24h. The apoptotic cells were determined by PI/Annexin V staining and flow cytometric analysis. Apoptotic ratio of cells treated by Imatinib together with TPL is significantly increased compared to cells treated by Imatinib alone (24.78±1.12 vs. 77.52±7.75, P<0.01). Next, the underlying mechanism was investigated. High expression of Hypoxia-inducible factor-1α (HIF-1α) has been shown to be related with bad clinical outcome in patients while Nrf2 is responsible for chemo-resistanct in cancer cells. Imatinib in combination with TPL could decrease Nrf2 and HIF-1α expression at protein and mRNA levels. Down-stream genes of Nrf2 e.g NQO1, GSR and HO-1 as well as target genes of HIF-1α e.g BNIP3, VEGF and CAIX are also down-regulated at mRNA level.

Conclusion

TPL could significantly increase the Imatinib-induced apoptotic ratio in K562/G cells through down-regulation of HIF-1α and Nrf2.

Disclosures:

No relevant conflicts of interest to declare.