Full-Length Immune Repertoire Reconstruction and Profiling at the Transcriptome Level Using Long-Read Sequencing.

BACKGROUND: Due to the diversity of the immune repertoire (IR), reconstructing full-length IR using traditional short-read sequencing has proven challenging. METHODS: A full-length IR sequencing (FLIRseq) work flow was developed with linear rolling circle amplification and nanopore sequencing. Its accuracy and quantification ability were verified by plasmid mixtures and commercial B-cell receptor/T-cell receptor sequencing (BCR/TCR-seq) based on short reads. IRs in tissues and the peripheral blood from 8 patients with acute lymphoblastic leukemia, 3 patients with allergic diseases, 4 patients with psoriasis, and 5 patients with prostate cancer were analyzed using FLIRseq. RESULTS: FLIRseq reads had lower mismatch rates and gap rates, and higher identify rates than nanopore reads (all P < 2.2 × -16). The relative quantification of components by FLIRseq was consistent with the actual quantification (P > 0.05). FLIRseq had superiority over BCR/TCR-seq, providing the long complementarity-determining region 3, B-cell isotype, and the rarely used V gene sequence. FLIRseq observed an increase in clonotype diversity (P < 0.05) and a decrease in the percentage of abnormal BCRs/TCRs in patients with leukemia in remission. For patients with allergic diseases or psoriasis, FLIRseq provided direct insights into V(D)J recombination and specific immunoglobulin classes. Compared with that in prostate cancer tissues, the full-length V segment of the biased T-cell receptor ß chain from lymphocytes in psoriatic tissues showed a more consistent AlphaFold2-predicted protein structure (P < 0.05). CONCLUSIONS: FLIRseq enables unbiased and comprehensive analyses of direct V(D)J recombination and immunoglobulin classes, thereby contributing to characterizing pathogenic mechanisms, monitoring minimal residual disease, and customizing adoptive cell therapy.

Case report: Difficult diagnosis of Mycobacterium tuberculosis infection in patients after allogeneic hematopoietic stem cell transplantation: two case reports and a literature review.

Background: Mycobacterium tuberculosis (MTB) is a relatively infrequent infection encountered during hematopoietic stem-cell transplantation (HSCT). The identification of MTB following HSCT remains a complex task, with delayed detection and misdiagnosis potentially resulting in unfavorable outcomes. Metagenomic next-generation sequencing (mNGS) represents a novel, highly sensitive, and rapid diagnostic tool in clinical settings for discerning intricate infections and detecting exceedingly rare pathogens. Methods: With the aid of mNGS, we diagnosed MTB in the lymph nodes and lungs of two patients with hematological diseases following allogeneic peripheral blood hematopoietic stem cell transplantation. Both patients presented with a fever, localized symptoms, and clinical signs. Following inconclusive results from routine tests, impractical biopsy procedures, and unsuccessful responses to empirical treatments, mNGS was employed as a final recourse, revealing DNA fragments of MTB in blood samples. Results: The diagnoses were ultimately confirmed in conjunction with additional clinical evidence. The application of mNGS in MTB cases after allogeneic HSCT has rarely been reported. The mNGS technique can provide a prompt and highly sensitive indication leading to the definitive diagnosis of MTB in complex post-transplant scenarios.

Case Report: CD19 CAR T-cell therapy following autologous stem cell transplantation: a successful treatment for R/R CD20-negative transformed follicular lymphoma with TP53 mutation.

Background: Follicular lymphoma (FL), a common indolent B-cell lymphoma, has the potential to transform into an aggressive lymphoma, such as diffuse large B-cell lymphoma (DLBCL). The outcome of patients with transformed follicular lymphoma (tFL) is poor, especially in patients with transformed lymphoma after chemotherapy and patients with progression within 24 months (POD24). Chimeric antigen receptor (CAR) T-cell therapy combined with autologous stem cell transplantation (ASCT) has promising antitumor efficacy. Case presentation: Here, we described a 39-year-old male patient who was initially diagnosed with FL that transformed into DLBCL with POD24, CD20 negativity, TP53 mutation, and a bulky mass after 3 lines of therapy, all of which were adverse prognostic factors. We applied a combination approach: CD19 CAR T-cell infusion following ASCT. Ibrutinib was administered continuously to enhance efficacy, DHAP was administered as a salvage chemotherapy, and ICE was administered as a bridging regimen. The patient underwent BEAM conditioning on days -7~ -1, a total of 3.8 × 106/kg CD34+ stem cells were infused on days 01~02, and a total of 108 CAR T cells (relmacabtagene autoleucel, relma-cel, JWCAR029) were infused on day 03. The patient experienced grade 2 cytokine release syndrome (CRS), manifesting as fever and hypotension according to institutional standards. There was no immune effector cell-associated neurotoxicity syndrome (ICANS) after CAR T-cell infusion. Finally, the patient achieved CMR at +1 month, which has been maintained without any other adverse effects. Conclusion: This case highlights the amazing efficacy of CD19 CAR T-cell therapy following ASCT for R/R tFL, thus providing new insight on therapeutic strategies for the future.

Donor-derived cytomegalovirus-cytotoxic T lymphocytes and leflunomide successfully control refractory cytomegalovirus infections and disease of multiple sites after allogeneic-hematopoietic stem cell transplantation: A case report.

Drug-resistant cytomegalovirus (CMV) infection after hematopoietic stem cell transplantation (HSCT) often leads to morbidity and mortality. Several studies have shown that CMV-cytotoxic T lymphocytes (CTLs) can overcome drug-resistant CMV infection, but still many questions remain unanswered. Here, we present a case of refractory CMV infection after allogeneic HSCT (allo-HSCT). Donor-derived CMV-CTLs failed to eliminate the virus in unique peripheral blood on the first application, when 70 mg methylprednisolone (MP) was taken per day. After a second attempt with a combination of 8 mg MP with leflunomide, a complete and persisting clearance of all involved sites, including peripheral blood, urinary system, leptomeninges, and retina, was achieved. To summarize, intravenous infusion of CTLs can eliminate CMV in the oculi and central nervous system (CNS), and a low dosage of 8 mg MP has no interaction with CMV-CTLs.

Clinical Characteristics and Outcome Analysis for HLA Loss Patients Following Partially Mismatched Related Donor Transplantation Using HLA Chimerism for Loss of Heterozygosity Analysis by Next-Generation Sequencing.

Genomic loss of mismatched human leukocyte antigen (HLA loss) is one of the most vital immune escape mechanisms of leukemic cells after allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, the methods currently used for HLA loss analysis have some shortcomings. Limited literature has been published, especially in lymphoid malignancies. This study aims to evaluate the incidences, risk factors of HLA loss, and clinical outcomes of HLA loss patients. In all, 160 patients undergoing partially mismatched related donor (MMRD) transplantation from 18 centers in China were selected for HLA loss analysis with the next-generation sequencing (NGS)-based method, which was validated by HLA-KMR. Variables of the prognostic risk factors for HLA loss or HLA loss-related relapse were identified with the logistic regression or the Fine and Gray regression model. An HLA loss detection system, HLA-CLN [HLA chimerism for loss of heterozygosity (LOH) analysis by NGS], was successfully developed. Forty (25.0%) patients with HLA loss were reported, including 27 with myeloid and 13 with lymphoid malignancies. Surprisingly, 6 of those 40 patients did not relapse. The 2-year cumulative incidences of HLA loss (22.7% vs 22.0%, P = 0.731) and HLA loss-related relapse (18.4% vs 20.0%, P = 0.616) were similar between patients with myeloid and lymphoid malignancies. The number of HLA mismatches (5/10 vs <5/10) was significantly associated with HLA loss in the whole cohort [odds ratio (OR): 3.15, P = 0.021] and patients with myeloid malignancies (OR: 3.94, P = 0.021). A higher refined-disease risk index (OR: 6.91, P = 0.033) and donor-recipient ABO incompatibility (OR: 4.58, P = 0.057) contributed to HLA loss in lymphoid malignancies. To sum up, HLA-CLN could overcome the limitations of HLA-KMR and achieve a better HLA coverage for more patients. The clinical characteristics and outcomes were similar in patients with HLA loss between myeloid and lymphoid malignancies. In addition, the results suggested that a patient with HLA loss might not always relapse.

Leukemia With TCF3-ZNF384 Rearrangement as a Distinct Subtype of Disease With Distinct Treatments: Perspectives From A Case Report and Literature Review.

BACKGROUND: ZNF384 rearrangements are found in 5-10% of B-cell acute lymphoblastic leukemia (B-ALL) and 48% of B cell/myeloid mixed phenotype acute leukemia (B/M MPAL). ZNF384-rearranged B-ALL is prone to lineage conversion after chemotherapy. TCF3 is the second most common rearrangement partner of ZNF384 in B-ALL (27.5%) and the most common partner in B/M MPAL (53.3%). TCF3-ZNF384 fusion is related to a poor steroid response and a high frequency of relapse. It is mostly reported in children and adolescents but rarely seen in adults. PATIENTS AND METHODS: Here, we report a rare case of adult common B-ALL with TCF3-ZNF384 fusion in which the patient relapsed after one cycle of consolidation chemotherapy. Relapsed leukemia cells from the bone marrow were cultured for 72 hours ex vivo, and a panel of 156 kinds of cytotoxic drugs, targeted therapy drugs, combination chemotherapy drugs, etc., was used for sensitivity screening. The literature on TCF3-ZNF384 fusion was reviewed, and reported cases with TCF3-ZNF384 fusion were summarized. Clinical characteristics were compared between B-ALL and MPAL with TCF3-ZNF384 fusion. RESULTS: The relapsed lymphoblasts showed moderate sensitivity to both acute myelocytic leukemia (AML) - and acute lymphoblastic leukemia (ALL)-directed combination chemotherapy schemes, as well as to multiple targeted therapeutic drugs. The hyper-CVAD (B) scheme showed synergistic effects with multiple targeted compounds and had the highest sensitivity. The patient chose the hyper-CVAD (B) scheme combined with sorafenib and achieved complete remission (CR), then consolidated with myeloid-directed homoharringtonine+cytarabine+daunorubicin (HAD) scheme and gained molecular CR. By reviewing the literature, we found that both the genomic landscapes and gene expression profiles of ZNF384-rearranged B-ALL and MPAL are similar and that both diseases have lineage plasticity. The gene expression profile in TCF3-ZNF384-positive patients shows enrichment of hematopoietic stem cell features. No significant differences in clinical characteristics were found between TCF3-ZNF384-positive ALL and MPAL. CONCLUSION: TCF3-ZNF384-positive leukemia may be a distinct subtype of leukemia regardless of immunophenotype. Considering the frequent lineage switches and sensitivity to both ALL- and AML-directed schemes, a uniform strategy directed at both lymphoid and myeloid lineages or at hematopoietic stem cells may be better for TCF3-ZNF384-positive leukemia. Small molecule targeted therapies may be promising treatment options and deserve further investigation.

Myeloid sarcoma of the colon as initial presentation in acute promyelocytic leukemia: A case report and review of the literature.

BACKGROUND: Myeloid sarcoma (MS) rarely occurs in acute promyelocytic leukemia (APL) at onset, but it can develop in relapse cases, especially after APL treated with all-trans retinoic acid (ATRA). Therefore little is known about the clinical features and suitable treatment for APL related MS due to the rarity of the disease, although this may be different from the treatment and prognosis of MS in the relapse stage. To our best knowledge, this is the second case report of APL initial presentation as colon MS. CASE SUMMARY: A 77-year-old woman complained of intermittent right lower abdominal pain, black stool, and difficult defecation for 2 mo. Physical examination showed diffuse tenderness during deep palpation and an anemic appearance. Laboratory findings showed positivity for fecal occult blood testing; white blood cell count: 3.84 × 109/L; hemoglobin: 105 g/L; platelet count: 174 × 109/L; and negativity for tumor markers. Abdominal enhanced computed tomography showed a space occupying lesion in the colon (1.9 cm). Fibrocolonoscopy revealed a polypoid and ulcerated mass measuring 2.5 cm. The tumor was removed. To our surprise, MS was confirmed by immunohistochemistry. PML/RARα fusion gene was detected in colon specimens by fluorescent in situ hybridization and real-time reverse transcription polymerase chain reaction, which was consistent with the bone marrow. She was diagnosed as having APL related MS. A smooth and unobstructed intestinal wall was found by fibrocolonoscopy, and continuous molecular remission was confirmed in both the bone marrow and colon after four courses of ATRA + arsenic trioxide (ATO). ATRA + ATO showed a favorable therapeutic response for both APL and MS. CONCLUSION: Early use of ATRA can benefit APL patients, regardless of whether MS is the first or recurrent manifestation.

A single-molecule van der Waals compass.

Single-molecule imaging is challenging but highly beneficial for investigating intermolecular interactions at the molecular level1-6. Van der Waals interactions at the sub-nanometre scale strongly influence various molecular behaviours under confinement conditions7-11. Inspired by the traditional compass12, here we use a para-xylene molecule as a rotating pointer to detect the host-guest van der Waals interactions in the straight channel of the MFI-type zeolite framework. We use integrated differential phase contrast scanning transmission electron microscopy13-15 to achieve real-space imaging of a single para-xylene molecule in each channel. A good correlation between the orientation of the single-molecule pointer and the atomic structure of the channel is established by combining the results of calculations and imaging studies. The orientations of para-xylene help us to identify changes in the van der Waals interactions, which are related to the channel geometry in both spatial and temporal dimensions. This work not only provides a visible and sensitive means to investigate host-guest van der Waals interactions in porous materials at the molecular level, but also encourages the further study of other single-molecule behaviours using electron microscopy techniques.

Atomic Spatial and Temporal Imaging of Local Structures and Light Elements inside Zeolite Frameworks.

Identifying the atomic structures of porous materials in spatial and temporal dimensions by (scanning) transmission electron microscope ((S)TEM) is significant for their wide applications in catalysis, separation and energy storage. However, the sensitivity of materials to electron beams made it difficult to reduce the electron damage to specimens while maintaining the resolution and signal-to-noise ratio. It is therefore still challenging to capture multiple images of the same area in one crystal to image the temporal changes of lattices. Usings integrated differential phase contrast (iDPC) STEM, atomic-resolution imaging of beam-sensitive zeolite frameworks is achieved with an ultralow dose of 40 e- Å-2 , 2-3 orders of magnitude lower than that of conventional STEM. Based on the iDPC technique, not only the atomic 3D architecture of ZSM-5 crystals but also the changes of frameworks are observed during in situ experiments. Local structures and light-element aromatics in ZSM-5 crystals can also be revealed directly under iDPC-STEM. These results provided not only an efficient tool to image beam-sensitive materials with ultralow beam current but also a new strategy to observe and investigate the hydrocarbon pools in zeolite catalysts at the single-molecule scale.

Transport Phenomena in Zeolites in View of Graph Theory and Pseudo-Phase Transition.

Transport phenomena play an essential role in catalysis. While zeolite catalysis is widely applied in industrial chemical processes, its efficiency is often limited by the transport rate in the micropores of the zeolite. Experimental and theoretical methods are useful for understanding the transport phenomena on multiscale levels. Traditional diffusion models usually use a linear driving force and an isotropic continuum medium, such that transport in a hierarchical catalyst structure and the occurrence of nonlinear deactivation cannot be well understood. Due to the presence of spatial confinement and an ordered structure, some aspects of the transport in a zeolite cannot be regarded as continuum phenomena and discrete models are being developed to explain these. Graph theory and small-world networks are powerful tools that have allowed pseudo-phase transition phenomena and other nontrivial relationships to be clearly revealed. Discrete models that include graph theory can build a bridge between microscopic quantum physics and macroscopic catalyst engineering in both the space and time scales. For a fuller understanding of transport phenomena in diverse fields, several theoretical methods need to be combined for a comprehensive multiscale analysis.

Highly selective conversion of methanol to propylene: design of an MFI zeolite with selective blockage of (010) surfaces.

As an important catalyst of methanol-to-propylene (MTP) conversion, the ZSM-5 zeolite has an anisotropic diffusion path and a large pore size, resulting in the formation of undesirable heavy aromatic by-products. Herein, we developed a surface-specific silica deposition method to block straight channels of nanosized ZSM-5 crystals selectively. By such a coating method, we can selectively suppress the yield of aromatics from the original 13% to 2.4% at 100% conversion of methanol. Trapped hydrocarbon pool species are directly confirmed by aberration-corrected S/TEM for the first time. Such a method of trapping and restricting hydrocarbon pool species in a multiscale zeolite with 10-membered rings would significantly increase its catalytic efficiency and olefin diffusion. Moreover, this provides new methodologies for zeolite structure construction and will be greatly beneficial for the industrial MTP process.

Development, Function, and Clinical Significance of Plasmacytoid Dendritic Cells in Chronic Myeloid Leukemia.

Plasmacytoid dendritic cells (pDC) are the main producers of a key T-cell-stimulatory cytokine, IFNα, and critical regulators of antiviral immunity. Chronic myeloid leukemia (CML) is caused by BCR-ABL, which is an oncogenic tyrosine kinase that can be effectively inhibited with ABL-selective tyrosine kinase inhibitors (TKI). BCR-ABL-induced suppression of the transcription factor interferon regulatory factor 8 was previously proposed to block pDC development and compromise immune surveillance in CML. Here, we demonstrate that pDCs in newly diagnosed CML (CML-pDC) develop quantitatively normal and are frequently positive for the costimulatory antigen CD86. They originate from low-level BCR-ABL-expressing precursors. CML-pDCs also retain their competence to maturate and to secrete IFN. RNA sequencing reveals a strong inflammatory gene expression signature in CML-pDCs. Patients with high CML-pDC counts at diagnosis achieve inferior rates of deep molecular remission (MR) under nilotinib, unless nilotinib therapy is combined with IFN, which strongly suppresses circulating pDC counts. Although most pDCs are BCR-ABL-negative in MR, a substantial proportion of BCR-ABL + CML-pDCs persists under TKI treatment. This could be of relevance, because CML-pDCs elicit CD8+ T cells, which protect wild-type mice from CML. Together, pDCs are identified as novel functional DC population in CML, regulating antileukemic immunity and treatment outcome in CML.Significance: CML-pDC originates from low-level BCR-ABL expressing stem cells into a functional immunogenic DC-population regulating antileukemic immunity and treatment outcome in CML. Cancer Res; 78(21); 6223-34. ©2018 AACR.

Analyzing transfer properties of zeolites using small-world networks.

Hierarchical structures bring efficiency to many processes, including metabolism, plant growth, and even social networks. In society, connectivity among people results in a hierarchical network with a superior efficiency for information transfer. These networks are known as small-world networks. Although the number of people known by a single person is insignificant compared with the total number of people in a society, a small number of long-range connections can bring extremely high information transfer efficiency to the social network. This is the key property of the small-world network. By modeling zeolite structures as small-world networks and regarding vacancies and cracks as long-range connections, we managed to quantify efficiency in hierarchical zeolite structures. We showed the influence of cracks and vacancies on the transfer phenomenon in zeolite structures. By adding 6% vacancies into a perfect 3D zeolite structure, we obtained a 30% equivalent volume reduction in zeolite crystals. This approach might result in new methodology for quantifying zeolite transfer and broaden horizons for zeolite structure design.

Carbon nanotube bundles with tensile strength over 80 GPa.

Carbon nanotubes (CNTs) are one of the strongest known materials. When assembled into fibres, however, their strength becomes impaired by defects, impurities, random orientations and discontinuous lengths. Fabricating CNT fibres with strength reaching that of a single CNT has been an enduring challenge. Here, we demonstrate the fabrication of CNT bundles (CNTBs) that are centimetres long with tensile strength over 80 GPa using ultralong defect-free CNTs. The tensile strength of CNTBs is controlled by the Daniels effect owing to the non-uniformity of the initial strains in the components. We propose a synchronous tightening and relaxing strategy to release these non-uniform initial strains. The fabricated CNTBs, consisting of a large number of components with parallel alignment, defect-free structures, continuous lengths and uniform initial strains, exhibit a tensile strength of 80 GPa (corresponding to an engineering tensile strength of 43 GPa), which is far higher than that of any other strong fibre.

[Investigation on resource and quality assessment of Cibotii Rhizoma].

The study is aimed to understand the resource and the current situation of the use of Cibotii Rhizoma and provide the basis for protecting and utilization. The method of literature survey, field survey and quality assessment were applied in the study. The results showed that all the Cibotii Rhizoma came from wild resource and was mainly founded in Fujian, Guangxi, Guizhou, Yunnan, Guangdong, Hunan, Jiangxi, Sichuan, Chongqing, Zhejiang, etc. It contains over 5 000 000 kg in the area which total is about 7 000 hm2. The annual output is over 850 000 kg. At present, there is no cultivated resources. Based on the investigation and market sampling analysis from various regions, the results showed that the quality of the collected crude drugs conformed with the regulations of the Chinese pharmacopoeia. However the qualification rate of decoction pieces of Cibotii Rhizoma in market was only 56.4%. At present, the resource of Cibotii Rhizoma could meet the needs of medinal uses. It is important to protect the wild resource which is less and less because of the environmental factors. It also need to make a standard of processing method to ensure the safety, and solve quality problem of the decoction pieces.

[Expression of death-associated protein kinase gene and methylation status of promoter region in acute leukemia].

This study was purpose to investigate the expression of death-associated protein kinase (DAPK) gene in acute leukemia (AL) patients and the methylation status of its promoter region through experiments of DAPK methylation and expression, and to analyze the relation between them. The expression of DAPK gene in leukemia cells and normal bone marrow cells was detected by RT-PCR; the methylation status of DAPK gene promoter region in cells from AL patients and leukemia cell lines HL-60 and U937 was detected by nested methylation specific PCR (n-MSP); 2 randomly primers selected from randomly amplified products of second round nMS-PCR were cloned and sequenced in professional company. The results showed that the DAPK gene expressed in bone marrow specimens of all 10 normal controls, with average value of expression 0.92 ± 0.18, while the average value of DAPK expression in bone marrow specimens of AL patients was 0.61 ± 0.40 which was lower than that in normal controls (P < 0.05). The low or deletion of DAPK mRNA expression were found in bone marrow specimens of 9/17 (52.94%) cases of ALL and 42/102 (41.18%) cases of AML. The cell line U937 showed normal expression of DAPK gene, while cell line HL-60 showed the expression detection of DAPK gene. The methylation of DAPK promoter region existed in 33 out of bone marrow specimens of 102 AML patients and in 8 out of bone marrow specimens of 17 ALL patients, the methylation rates were 32.4% (33/102) and 47% respectively. The DAPK promoter region in bone marrow of 7 normal controls was unmethylated, while DAPK promoter region in U937 cells and HL-60 cells were unmethylated and methylated respectively. The DAPK mRNA expression in ALL and AML patients significantly negatively correlated with the methylation of its promoter region (r = -0.855, P < 0.05, in AML patients and r = -0.343, P < 0.05, in AML patients) suggesting the close relationship between them. It is concluded that the methylation of DAPK gene promoter region relates with abnormal expression or detection of DAPK mRNA in AL patients.

Investigation of the mechanism involved in the As2O3-regulated decrease in MDR1 expression in leukemia cells.

Arsenic trioxide (As2O3) inhibits the expression of P-glycoprotein (P-gp) in leukemia cells; however, the mechanism behind this inhibition is unclear. The present study aimed to explore the effect of As2O3 on the expression and regulation of P-gp in leukemia cells, and elucidate the mechanism of the reversal of drug resistance. In the present study, electrophoretic mobility shift assay results indicated that p65 binds to the NF-κB binding site of MDR1, specifically in K562/D cells. Expression of p65 and phosphorylated IκB was reduced, while the expression of IκB was increased in K562/D cells treated with As2O3. The activity of luciferase increased up to 9-fold with 40 ng/ml TNF-α, and it was suppressed by ~25% following treatment with 1 µM As2O3. These findings suggest that As2O3 reverses the P-gp-induced drug resistance of leukemia cells through the NF-κB pathway. As2O3 may inhibit the activity of phosphorylase to inhibit IκB phosphorylation, thereby inhibiting NF-κB activity and MDR1 gene expression, leading to reversal of drug resistance.

Adaptive secretion of granulocyte-macrophage colony-stimulating factor (GM-CSF) mediates imatinib and nilotinib resistance in BCR/ABL+ progenitors via JAK-2/STAT-5 pathway activation.

Overcoming imatinib mesylate (IM) resistance and disease persistence in patients with chronic myeloid leukemia (CML) is of considerable importance to the issue of potential cure. Here we asked whether autocrine signaling contributes to survival of BCR/ABL+ cells in the presence of IM and nilotinib (NI; AMN107), a novel, more selective Abl inhibitor. Conditioned media (CM) of IM-resistant LAMA84 cell clones (R-CM) was found to substantially protect IM-naive LAMA cells and primary CML progenitors from IM- or NI-induced cell death. This was due to an increased secretion of the granulocyte-macrophage colony-stimulating factor (GM-CSF), which was identified as the causative factor mediating IM resistance in R-CM. GM-CSF elicited IM and NI drug resistance via a BCR/ABL-independent activation of the janus kinases 2 (JAK-2)/signal transducer and activator of transcription 5 (STAT-5) signaling pathway in GM-CSF receptor alpha receptor (CD116)-expressing cells, including primary CD34+/CD116+ GM progenitors (GMPs). Elevated mRNA and protein levels of GM-CSF were detected in IM-resistant patient samples, suggesting a contribution of GM-CSF secretion for IM and NI resistance in vivo. Importantly, inhibition of JAK-2 with AG490 abrogated GM-CSF-mediated STAT-5 phosphorylation and NI resistance in vitro. Together, adaptive autocrine secretion of GM-CSF mediates BCR/ABL-independent IM and NI resistance via activation of the antiapoptotic JAK-2/STAT-5 pathway. Inhibition of JAK-2 overcomes GM-CSF-induced IM and NI progenitor cell resistance, providing a rationale for the application of JAK-2 inhibitors to eradicate residual disease in CML.

FLT3-ITD-, but not BCR/ABL-transformed cells require concurrent Akt/mTor blockage to undergo apoptosis after histone deacetylase inhibitor treatment.

Leukemias are differentially sensitive to histone deacytelase inhibitor (HDI)-induced apoptosis, but molecular reasons for this remain unclear. We here show that BCR/ABL-, but not FMS-like tyrosine kinase 3 (FLT3)-internal tandem duplication (ITD)-transformed 32D cells or primary acute myeloid leukemia (AML) blasts undergo apoptosis after treatment with the HDI valproic acid (VPA) plus all-trans retinoic acid (VPA/ATRA). A particular VPA/ATRA responsiveness of Philadelphia chromosome-positive (Ph+) acute lymphatic leukemia (ALL) was confirmed in a therapy-refractory patient in vivo. HDI-stimulated apoptosis in Ph+ cells was caspase dependent, but independent from Akt pathway inhibition. Conversely, separate blockage of the Akt/mTor-signaling pathway was a prerequisite for overcoming apoptosis resistance to VPA/ATRA in FLT3-ITD cells, and primary AML blasts (n = 9). In conclusion, constitutive Akt activation causes apoptosis resistance to VPA/ATRA in AML, but not in Ph+ leukemia. This warrants the application of HDI-based therapies in poor-risk Ph+ ALL, and the use of Akt/mTor inhibitors to overcome HDI resistance in AML.

Interferon consensus sequence binding protein (ICSBP; IRF-8) antagonizes BCR/ABL and down-regulates bcl-2.

BCR/ABL is the causative genetic aberration in chronic myelogenous leukemia (CML). Mice lacking expression of the interferon (IFN) consensus sequence binding protein (ICSBP), an IFN gamma-inducible transcription factor of the interferon regulatory factor (IRF) family, develop a disease similar to human CML. Mounting evidence suggests a role for ICSBP in the pathogenesis of CML. However, the underlying mechanisms are largely unknown. By stable and conditional expression of ICSBP in wild-type and BCR/ABL-transformed 32D cells (32D/wt and 32D/BA), we found that ICSBP inhibited BCR/ABL-mediated leukemogenesis in vivo. Moreover, ICSBP also overrode BCR/ABL-mediated morphology changes, chemotherapy, and imatinib resistance, as well as BCR/ABL-induced repression of differentiation. Some of these ICSBP effects may be explained in part by an ICSBP-mediated repression of bcl-2, a major antiapoptotic target of BCR/ABL, on transcriptional and protein level. Using reporter gene assays and electrophoretic mobility shift assays we identified that the bcl-2 promoter activity was inhibited by ICSBP by way of a fragment containing 2 characteristic ICSBP-responsive elements. An inverse correlation between ICSBP and bcl-2 expression was confirmed in vivo. Collectively, our findings suggest that ICSBP antagonizes BCR/ABL by down-regulation of bcl-2 and implicates a central role for ICSBP in the pathogenesis of CML, as well as a therapeutic target to overcome drug resistance in bcl-2-dependent tumors.