Moyamoya disease presenting with symptomatic ischemic stroke during new-generation tyrosine kinase inhibitor treatment: two illustrative cases.

Tyrosine kinase inhibitors (TKIs) have been widely used to treat chronic myeloid leukemia. Nilotinib and ponatinib, which are second- and third-generation TKIs, have been reported to cause cerebrovascular arterial complications. Here, we present two cases of moyamoya disease presenting with symptomatic ischemic stroke during new-generation TKI treatment. We judged that new-generation TKI treatment was a factor in symptomatic ischemic stroke of unknown moyamoya disease in both cases. Noninvasive examinations using magnetic resonance imaging or carotid ultrasonography should be performed before and during new-generation TKI treatment in order to prevent symptomatic ischemic stroke.

Acute Tubulointerstitial Nephritis in Rosai-Dorfman Disease Mimicking IgG4-related Disease.

Rosai-Dorfman-Destombes disease (RDD) is a non-Langerhans cell histiocytosis characterized by the accumulation of histiocytes inside the lymph nodes or extranodally. The association between RDD and IgG4-related disease (IgG4-RD) is discussed. We herein report a case of RDD manifesting as acute tubulointerstitial nephritis mimicking IgG4-RD. The first renal biopsy showed severe tubulointerstitial nephritis with infiltration of S100-positive histiocytes and IgG4-positive plasma cells; storiform fibrosis and obliterative phlebitis were not confirmed. After prednisolone therapy, IgG4-positive cells and S100-positive histiocytes were decreased, but the IgG4/IgG ratio increased despite clinical improvement. These findings indicated extranodal RDD in the kidney presenting as tubulointerstitial nephritis.

Low-dose dasatinib in older patients with chronic myeloid leukaemia in chronic phase (DAVLEC): a single-arm, multicentre, phase 2 trial.

BACKGROUND: BCR-ABL1 tyrosine kinase inhibitors (TKIs) are commonly initiated in older patients with chronic myeloid leukaemia in the chronic phase at standard doses. However, because of their safety profile in this population, appropriate therapy has not been established. We aimed to investigate whether a lower than standard dose of dasatinib was an appropriate therapy for older patients with chronic myeloid leukaemia in the chronic phase. METHODS: DAsatinib, Very Low-dose, for Elderly CML-CP patients (DAVLEC) was a multicentre, single-arm, phase 2 trial done in 25 Japanese hospitals. We enrolled patients older than 70 years with newly diagnosed chronic myeloid leukaemia in the chronic phase, ECOG performance status 0-2, and no previous treatment for CML other than hydroxyurea within 4 weeks. Second-generation TKI dasatinib was given orally at a starting dose of 20% of the standard dose (20 mg/day). If the treatment was assessed as optimal response at 3 months, 6 months, and 9 months and adverse events were grade 2 or better (according to the NCI Common Toxicity Criteria v 4.0), the same dose was continued. If response was suboptimal and adverse events were grade 2 or better, the dose was increased by 20 mg/day. Once a dose reduction had been made because of a grade 3 or worse adverse event, there were no further dose increases. Treatment was discontinued if assessed as failure (disease progression to the accelerated phase or acute phase). The primary endpoint was the achievement of major molecular response at 12 months, assessed using a per-protocol analysis. This trial is registered at with the UMIN clinical trial registry, UMIN000024548, and has completed its planned observation period. FINDINGS: Between Nov 1, 2016, and Oct 30, 2019, 52 patients received first-line dasatinib therapy at 20 mg/day. The median age at diagnosis was 77·5 years (73·5-83·0). 35 (67%) patients were male and 17 (33%) were female. 31 (60%) of 52 patients reached major molecular response at 12 months (one-sided 95% CI 48-71), with a median follow-up of 366 days (IQR 353-372). Grade 3-4 adverse events were reported in 12 (23%) patients. Neutropenia was the most frequent grade 3-4 adverse event, occurring in three (6%) patients. No treatment-related deaths were observed. INTERPRETATION: Low-dose dasatinib at 20mg/day is worthy of consideration as a starting dose for older patients with newly diagnosed chronic myeloid leukaemia in the chronic phase. However, this dose needs to be further studied in a larger cohort and with a more ethnically diverse population. FUNDING: Bristol-Myers Squibb.

[Successful surgical resection of rectal cancer in a patient with relapsed hairy cell leukemia under interferon-α treatment].

An 83-year-old man was diagnosed with hairy cell leukemia (HCL). He was treated with cladribine and achieved partial remission. However, pancytopenia due to HCL bone marrow involvement progressed slowly. Nine years later, he developed rectal cancer. Prior to the surgery, endoscopy-assisted submucosal ink injection was performed to identify the area of lower intestinal lesions. The following day, he developed septic peritonitis with shock status, perhaps due to his neutropenia and ink injection procedures. Surgical resection of the cancer was presumed unfeasible; therefore, radiation was performed. Several months later, bone marrow examination revealed HCL infiltration with reticulin fibrosis. Chemotherapy regimens with purine nucleoside analogs, which are the standard treatments for HCL, might accentuate the progression of his rectal cancer and enhance the development of severe infections. Therefore, interferon (IFN) -α was administered as an alternative therapy. Three months later, pancytopenia resolved, and bone marrow examination revealed a remarkable improvement in HCL infiltration and marrow fibrosis. With IFN-α therapy, the patient successfully underwent surgical resection of the rectal cancer. Using INF-α, a prompt recovery from pancytopenia might be expected even in a patient with advanced HCL, who requires surgical treatment for a concomitant cancer.

[18F-fluorodeoxyglucose-positron emission tomography imaging before and after treatment of chronic-phase chronic myeloid leukemia with tyrosine kinase inhibitors].

A 64-year-old man presented with abnormal imaging results on 18F-fluorodeoxyglucose-positron emission tomography (FDG-PET), showing moderately increased FDG-uptake in the entire bone marrow. Blood tests revealed leukocytosis, thrombocytosis, and increased lactate dehydrogenase levels. Furthermore, the neutrophil alkaline phosphatase score decreased. Bone marrow examination revealed marked hypercellularity of myeloid and megakaryocytic lineages without an excess of blasts. Cytogenetic analysis of the bone marrow demonstrated Philadelphia chromosome, and fluorescence in situ hybridization analysis was positive for BCR-ABL1 fusion genes. Thus, the patient was diagnosed with chronic myeloid leukemia (CML) in the chronic phase and tyrosine kinase inhibitor therapy with 100 mg of dasatinib daily was initiated. Complete cytogenetic response and a major molecular response were achieved at 3 and 12 months post-treatment, respectively. FDG-uptake values of the bone marrow remarkably decreased along with the remission status of the disease. FDG-PET images at pre- and post-treatment of CML are rarely compared, so we report this case as an important reference.

[A Case of Malignant Lymphoma with an Elevated Serum IgG4].

A man in his 60s was referred to our hospital for further examination of left hydronephrosis and renal dysfunction. An enhanced abdominal computed tomographic scan showed that the patient had chronic abdominal aortic dissection and a non-enhancing retroperitoneal soft tissue occupying the front of the abdominal aorta as well as the bilateral common iliac arteries. The left ureter was compressed by the soft tissue at the fourth lumbar level. No tumor markers were significantly elevated and idiopathic retroperitoneal fibrosis was suspected to be the cause. Before starting treatment, however, right hydronephrosis was newly observed. We placed bilateral ureteral stents and reviewed our diagnosis. Elevated serum IgG4 and accumulation of 18F-fluorodeoxyglucose in the soft tissue were the points at issue. To determine the diagnosis, we performed open wedge biopsy. Histopathological findings showed mainly fibrous connective tissue with lymphocytic infiltration, which was positive for CD10, CD20, and bcl-2. These findings indicated follicular lymphoma. Induction chemotherapy was performed with 6 cycles of rituximab/cyclophosphamide/vincristine/prednisolone. The soft tissue tumor shrank markedly and the patient has been free from bilateral ureteral stents.

Methotrexate-related lymphoproliferative disorder of the stomach in a patient with rheumatoid arthritis: a case of disease regression after methotrexate cessation.

We report the case of a 78-year-old woman with methotrexate-related gastric lymphoproliferative disorder (LPD). The patient had a history of rheumatoid arthritis (RA) and had been treated with methotrexate (MTX). Endoscopic examination revealed round elevated lesions in the stomach, and a biopsy specimen showed atypical lymphoid cell proliferation. Immunohistological study found these atypical cells to be positive for L-26 but not for CD3 or EBER. Therefore, we made a diagnosis of MTX-related LPD showing features of diffuse large B-cell lymphoma. Combined positron emission tomography-computed tomography (PET-CT) using 18F-fluorodeoxyglucose (FDG) showed increased avidity in the stomach in addition to slightly increased FDG-avidity in the mediastinum and left chest wall. We decided not to start chemotherapy but to discontinue administration of MTX, with follow-up using endoscopy and PET-CT. The endoscopic examinations after cessation of MTX demonstrated gradual regression of the elevated lesions. PET-CT 6 months after cessation showed no increased FDG avidity in the stomach. While disease regression was observed in the stomach, the other FDG-avid spots remained unchanged on PET-CT. Therefore, we performed chemotherapy as additional therapy. On PET-CT after chemotherapy, the FDG-avid spots remained unchanged for more than 1 year, and we eventually concluded that they were RA-related inflammatory lesions. In patients with MTX-related LPD, cessation of MTX may be a therapeutic option, but careful follow-up and chemotherapy in accordance with the clinical course are essential.

Abacavir, an anti-HIV-1 drug, targets TDP1-deficient adult T cell leukemia.

Adult T cell leukemia (ATL) is an aggressive T cell malignancy caused by human T cell leukemia virus type 1 (HTLV-1) and has a poor prognosis. We analyzed the cytotoxic effects of various nucleoside analog reverse transcriptase inhibitors (NRTIs) for HIV-1 on ATL cells and found that abacavir potently and selectively kills ATL cells. Although NRTIs have minimal genotoxicities on host cells, the therapeutic concentration of abacavir induced numerous DNA double-strand breaks (DSBs) in the chromosomal DNA of ATL cells. DSBs persisted over time in ATL cells but not in other cell lines, suggesting impaired DNA repair. We found that the reduced expression of tyrosyl-DNA phosphodiesterase 1 (TDP1), a repair enzyme, is attributable to the cytotoxic effect of abacavir on ATL cells. We also showed that TDP1 removes abacavir from DNA ends in vitro. These results suggest a model in which ATL cells with reduced TDP1 expression are unable to excise abacavir incorporated into genomic DNA, leading to irreparable DSBs. On the basis of the above mechanism, we propose abacavir as a promising chemotherapeutic agent for ATL.

Small molecules that inhibit Vif-induced degradation of APOBEC3G.

BACKGROUND: HIV-1 Vif is essential for virus replication in natural target cells such as T cells and macrophages. Vif recruits a ubiquitin ligase to degrade restrictive APOBEC3 proteins. APOBEC3G is one of the most potent retroviral restriction factors targeted by Vif and, as such, the Vif-APOBEC3G interaction has emerged as a promising HIV-1 therapeutic target. METHODS: 20,000 small molecules were used in live-cell screens for those that preserve EGFP-APOBEC3G fluorescence and luciferase-APOBEC3G luminescence in the presence of HIV-1 Vif. RESULTS: 2 compounds with similar core structures preserved APOBEC3G levels in the presence of Vif. 10 µM of compound restored APOBEC3G to levels sufficient for incorporation into vif-proficient virus particles and restriction of virus infectivity. Vif-dependent APOBEC3G polyubiquitination and general proteasomal activity were unaffected at the same concentration. CONCLUSIONS: The small molecules described here preserve APOBEC3G levels and activity in the presence of Vif. These molecules are starting points for further development as antiretrovirals.

CKIP-1 is an intrinsic negative regulator of T-cell activation through an interaction with CARMA1.

The transcription factor NF-κB plays a key regulatory role in lymphocyte activation and generation of immune response. Stimulation of T cell receptor (TCR) induces phosphorylation of CARMA1 by PKCθ, resulting in formation of CARMA1-Bcl10-MALT1 (CBM) complex at lipid rafts and subsequently leading to NF-κB activation. While many molecular events leading to NF-κB activation have been reported, it is less understood how this activation is negatively regulated. We performed a cell-based screening for negative regulators of TCR-mediated NF-κB activation, using mutagenesis and complementation cloning strategies. Here we show that casein kinase-2 interacting protein-1 (CKIP-1) suppresses PKCθ-CBM-NF-κB signaling. We found that CKIP-1 interacts with CARMA1 and competes with PKCθ for association. We further confirmed that a PH domain of CKIP-1 is required for association with CARMA1 and its inhibitory effect. CKIP-1 represses NF-κB activity in unstimulated cells, and inhibits NF-κB activation induced by stimulation with PMA or constitutively active PKCθ, but not by stimulation with TNFα. Interestingly, CKIP-1 does not inhibit NF-κB activation induced by CD3/CD28 costimulation, which caused dissociation of CKIP-1 from lipid rafts. These data suggest that CKIP-1 contributes maintenance of a resting state on NF-κB activity or prevents T cells from being activated by inadequate signaling. In conclusion, we demonstrate that CKIP-1 interacts with CARMA1 and has an inhibitory effect on PKCθ-CBM-NF-κB signaling.

Defining HIV-1 Vif residues that interact with CBFß by site-directed mutagenesis.

Vif is essential for HIV-1 replication in T cells and macrophages. Vif recruits a host ubiquitin ligase complex to promote proteasomal degradation of the APOBEC3 restriction factors by poly-ubiquitination. The cellular transcription cofactor CBFß is required for Vif function by stabilizing the Vif protein and promoting recruitment of a cellular Cullin5-RING ubiquitin ligase complex. Interaction between Vif and CBFß is a promising therapeutic target, but little is known about the interfacial residues. We now demonstrate that Vif conserved residues E88/W89 are crucial for CBFß binding. Substitution of E88/W89 to alanines impaired binding to CBFß, degradation of APOBEC3, and virus infectivity in the presence of APOBEC3 in single-cycle infection. In spreading infection, NL4-3 with Vif E88A/W89A mutation replicated comparably to wild-type virus in permissive CEM-SS cells, but not in multiple APOBEC3 expressing non-permissive CEM cells. These results support a model in which HIV-1 Vif residues E88/W89 may participate in binding CBFß.

APOBEC3B can impair genomic stability by inducing base substitutions in genomic DNA in human cells.

Human APOBEC3 proteins play pivotal roles in intracellular defense against viral infection by catalyzing deamination of cytidine residues, leading to base substitutions in viral DNA. Activation-induced cytidine deaminase (AID), another member of the APOBEC family, is capable of editing immunoglobulin (Ig) and non-Ig genes, and aberrant expression of AID leads to tumorigenesis. However, it remains unclear whether APOBEC3 (A3) proteins affect stability of human genome. Here we demonstrate that both A3A and A3B can induce base substitutions into human genome as AID can. A3B is highly expressed in several lymphoma cells and somatic mutations occur in some oncogenes of the cells highly expressing A3B. Furthermore, transfection of A3B gene into lymphoma cells induces base substitutions in cMYC gene. These data suggest that aberrant expression of A3B can evoke genomic instability by inducing base substitutions into human genome, which might lead to tumorigenesis in human cells.

HIV-1 viral infectivity factor interacts with TP53 to induce G2 cell cycle arrest and positively regulate viral replication.

Viral infectivity factor, an accessory protein encoded in the HIV-1 genome, induces G2 cell cycle arrest; however, the biological significance and mechanism(s) remain totally unclear. Here we demonstrate that the TP53 pathway is involved in Vif-mediated G2 cell cycle arrest. Vif enhances the stability and transcriptional activity of TP53 by blocking the MDM2-mediated ubiquitination and nuclear export of TP53. Furthermore, Vif causes G2 cell cycle arrest in a TP53-dependent manner. HXB2 Vif lacks these activities toward TP53 and cannot induce G2 cell cycle arrest. Using mutagenesis, we demonstrate that the critical residues for this function are located in the N-terminal region of Vif. Finally, we construct a mutant NL4-3 virus with an NL4-3/HXB2 chimeric Vif defective for the ability to induce cell cycle arrest and show that the mutant virus replicates less effectively than the wild-type NL4-3 virus in T cells expressing TP53. These data imply that Vif induces G2 cell cycle arrest through functional interaction with the TP53/MDM2 axis and that the G2 cell cycle arrest induced by Vif has a positive effect on HIV-1 replication. This report demonstrates the molecular mechanisms and the biological significance of Vif-mediated G2 cell cycle arrest for HIV-1 infection.

MDM2 is a novel E3 ligase for HIV-1 Vif.

The human immunodeficiency virus type 1 (HIV-1) Vif plays a crucial role in the viral life cycle by antagonizing a host restriction factor APOBEC3G (A3G). Vif interacts with A3G and induces its polyubiquitination and subsequent degradation via the formation of active ubiquitin ligase (E3) complex with Cullin5-ElonginB/C. Although Vif itself is also ubiquitinated and degraded rapidly in infected cells, precise roles and mechanisms of Vif ubiquitination are largely unknown. Here we report that MDM2, known as an E3 ligase for p53, is a novel E3 ligase for Vif and induces polyubiquitination and degradation of Vif. We also show the mechanisms by which MDM2 only targets Vif, but not A3G that binds to Vif. MDM2 reduces cellular Vif levels and reversely increases A3G levels, because the interaction between MDM2 and Vif precludes A3G from binding to Vif. Furthermore, we demonstrate that MDM2 negatively regulates HIV-1 replication in non-permissive target cells through Vif degradation. These data suggest that MDM2 is a regulator of HIV-1 replication and might be a novel therapeutic target for anti-HIV-1 drug.

Phosphorylation of APOBEC3G by protein kinase A regulates its interaction with HIV-1 Vif.

Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G, referred to here as A3G) is a potent antiretroviral host factor against human immunodeficiency virus type 1 (HIV-1). HIV-1 viral infectivity factor (Vif) counteracts A3G by promoting its degradation via the ubiquitin-proteasome pathway. Recent studies demonstrated that protein kinase A (PKA) phosphorylates activation-induced deaminase (AID), another member of the APOBEC3 family. A3G has two putative PKA phosphorylation residues. Here we show that PKA binds and specifically phosphorylates A3G at Thr32 in vitro and in vivo. This phosphorylation event reduces the binding of A3G to Vif and its subsequent ubiquitination and degradation, and thus promotes A3G antiviral activity. Computer-assisted structural modeling and mutagenesis studies suggest that the interaction between A3G Thr32 and Arg24 is crucial for interaction with Vif. These data imply that PKA-mediated phosphorylation of A3G can regulate the interaction between A3G and Vif.