Clinical factors for central nervous system progression and survival in primary vitreoretinal lymphoma.

Primary vitreoretinal lymphoma (PVRL) is a rare subtype of malignant lymphoma with a poor prognosis because of high frequency of central nervous system (CNS) progression. Identification of factors associated with CNS progression is essential to improve the prognosis of patients with PVRL. We conducted a retrospective study of 54 patients diagnosed with PVRL and treated at our hospital to identify factors associated with CNS progression and prognosis. All patients were treated with intravitreal methotrexate (MTX) injections in the affected eyes until lesion resolution. Twenty-four patients were treated with systemic administration of high-dose MTX (systemic HD-MTX) every other week for a total of five cycles following intravitreal MTX injection. Of 24 patients, 20 completed five cycles of systemic HD-MTX. The 5-year cumulative incidence of CNS progression and overall survival (OS) rate were 78.0% and 69.0% respectively. By univariate and multivariate analyses, bilateral disease and the detection of B-cell clonality confirmed by flow cytometric analysis were risk factors associated with CNS progression. Moreover, systemic HD-MTX completion reduced the risk of CNS progression and was identified as a factor affecting OS. In this study, factors for CNS progression identified may potentially contribute to the optimized therapeutic stratification to improve the survival of patients with PVRL.

18q Deletion Syndrome Presenting with Late-Onset Combined Immunodeficiency.

Patients with chromosome 18q deletion syndrome generally experience hypogammaglobulinemia. Herein, we describe two patients with chromosome 18q deletion syndrome who presented with late-onset combined immune deficiency (LOCID), which has not been previously reported. Patient 1 was a 29-year-old male with 18q deletion syndrome, who was being managed for severe motor and intellectual disabilities at the Yamabiko Medical Welfare Center for 26 years. Although the patient had few infections, he developed Pneumocystis pneumonia at the age of 28. Patient 2, a 48-year-old female with intellectual disability and congenital malformations, was referred to Tokyo Medical and Dental University Hospital with abnormal bilateral lung shadows detected on her chest radiography. Computed tomography showed multiple lymphadenopathies and pneumonia. A lymph node biopsy of the inguinal region revealed granulomatous lymphadenitis, and a chromosomal examination revealed 18q deletion. Array-based genomic hybridization analysis revealed deletion at 18q21.32-q22.3 for patient 1 and at 18q21.33-qter for patient 2. Immune status work-up of the two patients revealed panhypogammaglobulinemia, decreased number of memory B cells and naïve CD4+ and/or CD8+ cells, reduced response on the carboxyfluorescein diacetate succinimidyl ester T-cell division test, and low levels of T-cell receptor recombination excision circles and Ig κ-deleting recombination excision circles. Consequently, both patients were diagnosed with LOCID. Although patients with 18q deletion syndrome generally experience humoral immunodeficiency, the disease can be further complicated by cell-mediated immunodeficiency, causing combined immunodeficiency. Therefore, patients with 18q deletion syndrome should be regularly tested for cellular/humoral immunocompetence.

Impact of genetic alterations on central nervous system progression of primary vitreoretinal lymphoma.

Primary vitreoretinal lymphoma (PVRL) is a rare malignant lymphoma subtype with an unfavorable prognosis due to frequent central nervous system (CNS) progression. Thus, identifying factors associated with CNS progression is essential for improving the prognosis of PVRL patients. Accordingly, we conducted a comprehensive genetic analysis using archived vitreous humor samples of 36 PVRL patients diagnosed and treated at our institution and retrospectively examined the relationship between genetic alterations and CNS progression. Whole-exome sequencing (n = 2) and amplicon sequencing using a custom panel of 107 lymphomagenesis-related genes (n = 34) were performed to assess mutations and copy number alterations. The median number of pathogenic genetic alterations per case was 12 (range: 0- 22). Pathogenic genetic alterations of CDKN2A, MYD88, CDKN2B, PRDM1, PIM1, ETV6, CD79B, and IGLL5, as well as aberrant somatic hypermutations, were frequently detected. The frequency of ETV6 loss and PRDM1 alteration (mutation and loss) was 23% and 49%, respectively. Multivariate analysis revealed ETV6 loss (hazard ratio [HR]: 3.26, 95% confidence interval [CI]: 1.08-9.85) and PRDM1 alteration (HR: 2.52, 95% CI: 1.03-6.16) as candidate risk factors associated with CNS progression of PVRL. Moreover, these two genetic factors defined slow-, intermediate-, and rapid-progression groups (0, 1, and 2 factors, respectively), and the median period to CNS progression differed significantly among them (52 vs. 33 vs. 20 months, respectively). Our findings suggest that genetic factors predict the CNS progression of PVRL effectively, and the genetics-based CNS progression model might lead to stratification of treatment.

Impact of BK polyomavirus viremia on the outcomes of allogeneic hematopoietic stem cell transplantation.

Although it is known that BK polyomavirus (BKPyV) causes hemorrhagic cystitis (HC) after allogeneic hematopoietic stem cell transplantation (HSCT), the clinical significance of BKPyV viremia has not been fully evaluated. We retrospectively analyzed the results of quantitative polymerase chain reaction (PCR) evaluations for detecting BKPyV in the whole blood samples of patients undergoing allogeneic HSCT during the period from January 2010 to June 2020 at a single institute, Tokyo Medical and Dental University. BKPyV was detected in the blood of 28 of the 107 evaluated patients, and the cumulative incidence of was 27.9% (95%CI: 20.2-37.9%). HC due to BKPyV developed in four of the 28 patients with BKPyV viremia (14.3%) and in two of the 79 patients without it (2.5%; P < 0.05). BKPyV viremia itself did not affect the patients' post-transplant estimated glomerular filtration rate (eGFR), but BKPyV viremia with a high viral load was significantly associated with decreased eGFR values (P < 0.05). BKPyV viremia was also associated with significantly lower progression-free survival at 3 years (35.1% [95%CI: 17.8-53.1%] vs. 60.4% [95%CI: 48.4-70.5], P < 0.05). Our findings demonstrated that BKPyV viremia was associated with onset of HC, an early decline of renal function, and poorer survival after allogeneic HSCT. Further studies are needed to test these results and elucidate the mechanisms of renal dysfunction associated with BKPyV viremia.

Lymphoproliferative disorder risk after methotrexate treatment for rheumatoid arthritis.

Methotrexate (MTX)-associated lymphoproliferative disorder (MTX-LPD) is a troublesome problem in patients receiving MTX for rheumatoid arthritis (RA). However, its incidence, prognosis, and risk factors remain unclear. In this retrospective study, we evaluated the actual incidence, prognostic impact, and risk factors of MTX-LPD. Of the 986 patients with RA treated with MTX, 90 patients experienced 95 new malignancies (NMs), with LPD as the most frequent in 26 patients. The cumulative LPD incidences were 1.3% and 4.7% at 5 and 10 years after MTX initiation, respectively. Among the 24 patients who discontinued MTX after developing LPD, 15 showed sustained regression, without difference in overall survival between patients with LPD and without NM. Inflammatory markers and absolute lymphocyte counts were not useful for early LPD development detection, but most of the patients with LPD had persistently elevated erythrocyte sedimentation ratios. Regarding concomitant drugs, tacrolimus increased the risk only if patients were not receiving biological disease-modifying antirheumatic drugs (bDMARDs). bDMARDs did not increase the risk for any of the drugs or the number of classes used. The number of LPD cases was lower in patients with IL-6A even after a long period after MTX, although with no statistically significant difference. Thus, approximately 1 in 20 patients with RA developed MTX-LPD over the 10 years of MTX treatment, but it did not affect the survival of patients with RA. Tacrolimus increased the risk of developing LPD for certain patients and should be used with caution.

[Acute promyelocytic leukemia with asymptomatic cerebral hemorrhage].

A 43-year-old man presenting with oral bleeding was diagnosed with acute promyelocytic leukemia (APL). Induction chemotherapy consisting of all-trans retinoic acid and idarubicin was initiated, and disseminated intravascular coagulation (DIC) was treated with fresh frozen plasma and recombinant thrombomodulin infusions. The patient was free from neurological symptoms throughout the clinical course. However, cerebral hemorrhagic lesions were detected incidentally on magnetic resonance imaging performed to screen for leukemic central nervous system invasion at 2 weeks after treatment initiation. Imaging findings suggested subacute or later-phase cerebral hemorrhage. Platelet transfusions and other supportive care was provided. Serial imaging evaluations confirmed reduction of the hemorrhagic lesions. Hematological remission was achieved after induction chemotherapy, and no symptoms due to cerebral hemorrhage developed during the subsequent consolidation therapy. As patients with APL characteristically experience hemorrhagic events due to bleeding tendency caused by DIC, physicians should be aware of the possibility of asymptomatic cerebral hemorrhage in these patients.

[Malignant paraganglioma mimicking multiple myeloma].

We here present a 33-year-old woman who was referred to our hospital with a complaint of back pain and was found to have elevated IgG and hypercalcemia, as well as osteolytic lesions of pelvis and spines. 18F-FDG-PET/CT scan revealed numerous uptakes in the bones. An examination of the bone marrow revealed increased plasma cells (10.2%). Despite clinical similarities to multiple myeloma, any evidence of plasma cell clonal proliferation, including serum M-protein and light chain restriction, was not found. A reexamination of the bone marrow with a biopsy revealed the proliferation of abnormal cells with chromogranin A and synaptophysin expression but no expression of hematopoietic and epithelial cell markers. Based on these results together with extra-adrenal lesions, a diagnosis of malignant paraganglioma was made. Malignant paraganglioma is known to frequently cause bone metastasis and skeletal related events, whose clinical manifestations are similar to those of multiple myeloma. Since patients with osteolytic lesions, hypercalcemia, and hypergammaglobulinemia are likely to be referred to hematologists, malignant paraganglioma should be considered as a differential diagnosis.

[Perioperative management of laparoscopic cholecystectomy in a patient with paroxysmal nocturnal haemoglobinuria undergoing ravulizumab treatment].

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by hemolysis, thrombosis, and bone marrow failure. Infection, pregnancy, and surgical operation have the potential to evoke severe episodes of hemolysis and thrombosis. Therefore, the use of an antibody agent against complement component 5 (C5), eculizumab, one day before the operation is recommended. Ravulizumab is a newly approved long-acting antibody agent against C5. Thus, little is known about perioperative management with ravulizumab. We experienced a 43-year-old female patient who safely underwent laparoscopic cholecystectomy under ravulizumab treatment for PNH. Ravulizumab was administered one day before the operation. Laparoscopic cholecystectomy for cholelithiasis was performed under intravenous anesthesia, intermittent air compression of the lower extremities, and low pneumoperitoneum pressure. Additionally, heparin was administered, and the patient left the sickbed early without significant postoperative complications. Like eculizumab, complement inhibition by ravulizumab is also considered effective in the perioperative management of patients with PNH. However, close cooperation with surgeons and anesthesiologists and careful management based on clinical symptoms and laboratory data such as LDH, CH50, and D-dimer are essential.

[Impaired hematopoiesis due to copper deficiency in a hemodialysis patient supplemented with zinc].

This is a case of a 75-year-old man who was on maintenance hemodialysis for 10 years due to diabetic nephropathy and was prescribed polaprezinc due to a low serum zinc level (55 µg/dl) and dysgeusia. Three months after the polaprezinc treatment was initiated, the patient developed pancytopenia, which persisted even after the serum zinc level was normalized and medication was discontinued. He was referred to our institute so that the progression of pancytopenia could be assessed. A blood biochemical examination revealed a WBC count of 1,700/µl, Hb level of 8.9 g/dl, and Plt count of 9.5×104/µl. A bone marrow aspirate smear showed slight megaloblastic changes and ringed sideroblasts in addition to an elevated WT1 mRNA level (76 copies/µg RNA) in the peripheral blood. Although these findings mimicked those of myelodysplasia, low serum copper (<2 µg/dl) and ceruloplasmin levels (3 mg/dl) were suggestive of hematopoietic abnormalities due to zinc-induced copper deficiency. Treatment with cocoa, a compound generally known to be rich in copper, gradually improved the pancytopenia and dysplastic bone marrow histology. This case indicates that clinicians should consider the risk of zinc-induced copper deficiency and its complications when zinc supplementation is administered to patients with chronic kidney disease, particularly those undergoing hemodialysis.

c-Cbl and Cbl-b ligases mediate 17-allylaminodemethoxygeldanamycin-induced degradation of autophosphorylated Flt3 kinase with internal tandem duplication through the ubiquitin proteasome pathway.

The class III receptor-tyrosine kinase Flt3 regulates normal hematopoiesis. An internal tandem duplication (ITD) in the juxtamembrane domain of Flt3 (Flt3-ITD) contributes to transformation and is associated with poor prognosis in acute myeloid leukemia. Here, we demonstrate that, as compared with wild-type Flt3 (Flt3-WT), Flt3-ITD more rapidly undergoes degradation through the proteasomal and lysosomal pathways in model hematopoietic 32D cells and in human leukemic MV4-11 cells. The Hsp90 inhibitor 17-allylaminodemethoxygeldanamycin (17-AAG) preferentially induced the polyubiquitination and proteasomal degradation of Flt3-ITD autophosphorylated on Tyr-591 in these cells. The E3 ubiquitin ligases c-Cbl and to a lesser extent Cbl-b facilitated at least partly Lys-48-linked polyubiquitination of autophosphorylated Flt3-ITD when coexpressed in 293T cells. Moreover, c-Cbl and Cbl-b facilitated degradation of Flt3-ITD in 293T cells and significantly enhanced the 17-AAG-induced decline in autophosphorylated Flt3-ITD. The enhancement of Flt3-ITD degradation was also observed in 32D cells inducibly overexpressing c-Cbl or Cbl-b. Furthermore, overexpression of loss-of-function mutants of both c-Cbl (c-Cbl-R420Q) and Cbl-b (Cbl-b-C373A) together in 32D cells retarded the degradation of autophosphorylated Flt3-ITD and significantly inhibited the 17-AAG-induced degradation of Flt3-ITD to confer the resistance to cytotoxicity of 17-AAG on these cells. These results suggest that c-Cbl as well as Cbl-b may play important roles in Hsp90 inhibitor-induced degradation of Flt3-ITD through the ubiquitin proteasome system and in regulation of the basal expression level of Flt3-ITD in leukemic cells.

Challenges in the diagnosis and management of vitreoretinal lymphoma - Clinical and basic approaches.

Vitreoretinal lymphoma (VRL) is a subtype of diffuse large B-cell lymphoma and is sight- and life-threatening in the vast majority of patients. Lymphoma cells infiltrate the vitreous body and/or subretinal space and exhibit clinical signs of vitreous opacities and creamy white subretinal lesions. Although the intraocular signs can serve as clues to suspect VRL, they are nonspecific and may be misdiagnosed as uveitis. Histopathological evidence of malignant cells on vitreous biopsy, for instance, is the gold standard for diagnosis of VRL; however, cytological examination of the vitreous often results in a low success rate owing to the small quantity and poor quality of tissues and cells in the sample. Recent advancements in immunological, molecular, and gene analyses using intraocular samples have made it possible to accurately diagnose VRL. As for the management of VRL, local treatments with irradiation and/or intravitreal injections of anti-tumor agents (methotrexate or rituximab) are effective in suppressing intraocular VRL lesions. However, the effect of systemic chemotherapy, with or without brain irradiation, on preventing central nervous system involvements remains controversial. In this review article, we discuss the following concepts based on previous literature and our unpublished results: current ocular imaging examinations such as optical coherence tomography and fundus autofluorescence; immunological, molecular, and gene expression characterization of intraocular biopsies with special attention to flow cytometry; immunoglobulin gene rearrangement assays that use the polymerase chain reaction test; cytokine assays; gene mutations (MYD88, CD79B); and current local and systemic treatments of VRL.

Establishment and characterization of a new activated B-cell-like DLBCL cell line, TMD12.

We report the establishment of a novel activated B-cell-like (ABC) diffuse large B-cell lymphoma (DLBCL) cell line, designated as TMD12, from a patient with highly refractory DLBCL. ABC-DLBCL is a subtype with a relatively unfavorable prognosis that was originally categorized using gene expression profiling according to its cell of origin. TMD12 cells were isolated from the pleural effusion of the patient at relapse and passaged continuously in vitro for >4 years. The cells displayed cluster of differentiation (CD)19, CD20, CD22, CD38, human leukocyte antigen-DR isotype, and κ positivity and CD5, CD10, CD23, and λ negativity, as detected using flow cytometric analysis. The chromosomal karyotypic analysis, including the spectral karyotyping method, confirmed t(1;19)(q21:q13.1), del(6q23), gain of chromosome 18, and other abnormalities. Mutation analyses, including whole-exome sequencing, revealed that TMD12 cells harbored mutations in MYD88 and CD79B, indicating an ABC subtype. TMD12 cells exhibited chronic active B-cell receptor signaling and constitutive activation of the nuclear factor κB pathway, which is typically associated with sensitivity to a specific Bruton tyrosine kinase inhibitor, ibrutinib. Intriguingly, TMD12 cells displayed moderate resistance to ibrutinib and lacked activation of Janus kinase/signal transducers and activators of transcription 3 signaling, another hallmark of this DLBCL subtype. Treatment with an inhibitor against tumor progression locus 2 (TPL2), a multifunctional intracellular kinase that is activated particularly downstream of Toll-like receptors or MYD88 and IκB kinase α/ß (IKKα/ß), suppressed the proliferation of TMD12 cells, implying the possible involvement of the TPL2-p105 pathway in the tumorigenesis of ABC-DLBCL. Because only a limited number of ABC-DLBCL cell lines are currently available, TMD12 cells might provide a useful tool in the search for novel druggable targets for this intractable lymphoma.

Severe Motor Weakness Due to Disturbance in Peripheral Nerves Following Tisagenlecleucel Treatment.

BACKGROUND: Neurotoxicity is one of the dangerous complications of chimeric antigen receptor (CAR) T-cell therapy, while its pathophysiology remains to be fully understood. Motor weakness not associated with central nervous system (CNS) toxicity has rarely been reported after CAR T-cell therapy. CASE REPORT: A 42-year-old female with a refractory diffuse large B-cell lymphoma received tisagenlecleucel (tisa-cel) and developed cytokine release syndrome (CRS) on day 3. She was treated with tocilizumab and methylprednisolone, which resolved CRS promptly. On day 7, motor weakness in lower extremities appeared, and she gradually became unable to walk without showing any other symptoms attributed to CNS disturbances. Whereas dexamethasone and tocilizumab were ineffective, neuropathy improved after high dose chemotherapy followed by autologous stem cell transplantation. Nerve conduction study (NCS) in lower extremities showed a decline in compound muscle action potential amplitude along with worsening of motor weakness, which was restored after improvement of symptoms. Based on symptoms and NCS, her motor weakness was thought to be due to disturbance in peripheral nerves. CONCLUSION: This study reports a patient who developed severe motor weakness due to disturbance in peripheral nerves after tisa-cel therapy. Neurotoxicity of non-CNS origin should also be noted in CAR T-cell therapy.

An Antibody-Drug Conjugate That Selectively Targets Human Monocyte Progenitors for Anti-Cancer Therapy.

As hematopoietic progenitors supply a large number of blood cells, therapeutic strategies targeting hematopoietic progenitors are potentially beneficial to eliminate unwanted blood cells, such as leukemic cells and immune cells causing diseases. However, due to their pluripotency, targeting those cells may impair the production of multiple cell lineages, leading to serious side effects such as anemia and increased susceptibility to infection. To minimize those side effects, it is important to identify monopotent progenitors that give rise to a particular cell lineage. Monocytes and monocyte-derived macrophages play important roles in the development of inflammatory diseases and tumors. Recently, we identified human monocyte-restricted progenitors, namely, common monocyte progenitors and pre-monocytes, both of which express high levels of CD64, a well-known monocyte marker. Here, we introduce a dimeric pyrrolobenzodiazepine (dPBD)-conjugated anti-CD64 antibody (anti-CD64-dPBD) that selectively induces the apoptosis of proliferating human monocyte-restricted progenitors but not non-proliferating mature monocytes. Treatment with anti-CD64-dPBD did not affect other types of hematopoietic cells including hematopoietic stem and progenitor cells, neutrophils, lymphocytes and platelets, suggesting that its off-target effects are negligible. In line with these findings, treatment with anti-CD64-dPBD directly killed proliferating monocytic leukemia cells and prevented monocytic leukemia cell generation from bone marrow progenitors of chronic myelomonocytic leukemia patients in a patient-derived xenograft model. Furthermore, by depleting the source of monocytes, treatment with anti-CD64-dPBD ultimately eliminated tumor-associated macrophages and significantly reduced tumor size in humanized mice bearing solid tumors. Given the selective action of anti-CD64-dPBD on proliferating monocyte progenitors and monocytic leukemia cells, it should be a promising tool to target cancers and other monocyte-related inflammatory disorders with minimal side effects on other cell lineages.

Potent antitumor effects of cell-penetrating peptides targeting STAT3 axis.

To date, there are no inhibitors that directly and specifically target activated STAT3 and c-Myc in the clinic. Although peptide-based inhibitors can selectively block activated targets, their clinical usage is limited because of low cell penetration and/or serum stability. Here, we generated cell-penetrating acetylated (acet.) STAT3, c-Myc, and Gp130 targeting peptides by attaching phosphorothioated (PS) polymer backbone to peptides. The cell-penetrating peptides efficiently penetrated cells and inhibited activation of the intended targets and their downstream genes. Locally or systemically treating tumor-bearing mice with PS-acet.-STAT3 peptide at low concentrations effectively blocked STAT3 in vivo, resulting in significant antitumor effects in 2 human xenograft models. Moreover, PS-acet.-STAT3 peptide penetrated and activated splenic CD8+ T cells in vitro. Treating immune-competent mice bearing mouse melanoma with PS-acet.-STAT3 peptide inhibited STAT3 in tumor-infiltrating T cells, downregulating tumor-infiltrating CD4+ T regulatory cells while activating CD8+ T effector cells. Similarly, systemic injections of the cell-penetrating c-Myc and Gp130 peptides prevented pancreatic tumor growth and induced antitumor immune responses. Taken together, we have developed therapeutic peptides that effectively and specifically block challenging cancer targets, resulting in antitumor effects through both direct tumor cell killing and indirectly through antitumor immune responses.

Soluble Interleukin-2 Receptor Level at Diagnosis Predicts Prognosis of Patients With Follicular Lymphoma Irrespective of Initial Management Strategy.

BACKGROUND/AIM: Although various prognostic indices for follicular lymphoma (FL) have been proposed, they are designed specifically for patients requiring immediate therapy. We aimed to develop a new simple prognostic tool applicable for all patients with FL at diagnosis. MATERIALS AND METHODS: We retrospectively analyzed various clinical, pathological, and laboratory data, including soluble interleukin-2 receptor (sIL2R), from 140 patients with FL from two centers for their impact on prognosis. This study analyzed the impact of soluble interleukin-2 receptor (sIL2R) in order to develop a new simple prognostic tool applicable for all patients with FL at diagnosis. RESULTS: The initial management of these patients was watchful waiting (n=48) or immediate treatment (n=92). Event-free survival at 24 months predicted overall survival. When categorized into three groups according to the sIL2R levels at diagnosis, a very high sIL2R level identified about 20% of patients with a distinctively worse survival compared to the others. CONCLUSION: sIL2R is a very effective biomarker that can be easily applied in routine practice to predict survival for all patients with FL at diagnosis irrespective of initial management approach.

An effective cell-penetrating antibody delivery platform.

Despite their well-recognized success in the clinic, antibodies generally do not penetrate cellular membranes to target intracellular molecules, many of which underlie incurable diseases. Here we show that covalently conjugating phosphorothioated DNA oligonucleotides to antibodies enabled their efficient cellular internalization. Antibody cell penetration was partially mediated by membrane potential alteration. Moreover, without an antigen to bind, intracellular levels of the modified antibodies underwent cellular clearance, which involved efflux and lysosomal degradation, enabling detection of intended intracellular molecules as tested in fibroblasts, tumor cells, and T cells. This target-dependent cellular retention of modified antibodies extended to in vivo studies. Both local and systemic administrations of low doses of modified antibodies effectively inhibited intracellular targets, such as transcription factors Myc, interferon regulatory factor 4, and tyrosine-protein kinase SRC, and expression of their downstream genes in tumors, resulting in tumor cell apoptosis and tumor growth inhibition. This simple modification enables the use of antibodies to detect and modulate intracellular molecules in both cultured living cells and in whole animals, forming the foundation for a new paradigm for antibody-based research, diagnostics, and therapeutics.

Mechanisms for mTORC1 activation and synergistic induction of apoptosis by ruxolitinib and BH3 mimetics or autophagy inhibitors in JAK2-V617F-expressing leukemic cells including newly established PVTL-2.

The activated JAK2-V617F mutant is very frequently found in myeloproliferative neoplasms (MPNs), and its inhibitor ruxolitinib has been in clinical use, albeit with limited efficacies. Here, we examine the signaling mechanisms from JAK2-V617F and responses to ruxolitinib in JAK2-V617F-positive leukemic cell lines, including PVTL-2, newly established from a patient with post-MPN secondary acute myeloid leukemia, and the widely used model cell line HEL. We have found that ruxolitinib downregulated the mTORC1/S6K/4EBP1 pathway at least partly through inhibition of the STAT5/Pim-2 pathway with concomitant downregulation of c-Myc, MCL-1, and BCL-xL as well as induction of autophagy in these cells. Ruxolitinib very efficiently inhibited proliferation but only modestly induced apoptosis. However, inhibition of BCL-xL/BCL-2 by the BH3 mimetics ABT-737 and navitoclax or BCL-xL by A-1331852 induced caspase-dependent apoptosis involving activation of Bak and Bax synergistically with ruxolitinib in HEL cells. On the other hand, the putative pan-BH3 mimetic obatoclax as well as chloroquine and bafilomycin A1 inhibited autophagy at its late stage and induced apoptosis in PVTL-2 cells synergistically with ruxolitinib. The present study suggests that autophagy as well as the anti-apoptotic BCL-2 family members, regulated at least partly by the mTORC1 pathway downstream of STAT5/Pim-2, protects JAK2-V617F-positive leukemic cells from ruxolitinib-induced apoptosis depending on cell types and may contribute to development of new strategies against JAK2-V617F-positive neoplasms.

Acute myeloid leukemia with t(3;21)(q26.2;q22) developing following low-dose methotrexate therapy for rheumatoid arthritis and expressing two AML1/MDS1/EVI1 fusion proteins: A case report.

The t(3;21)(q26.2;q22) translocation is a rare chromosomal abnormality exhibited almost exclusively in therapy-related myelodysplastic syndrome/acute myeloid leukemia (t-MDS/AML) or in the blastic crisis phase of chronic myelogenous leukemia, which results in the fusion of the runt related transcription factor 1 (RUNX1, also called AML1) gene at 21q22 to the myelodysplasia syndrome 1 (MDS1)-ecotropic virus integration site 1 (EVI1) complex locus (MECOM) at 3q26.2, generating various fusion transcripts, including AML1/MDS1/EVI1 (AME). The present study examined the case of an 84-year-old Japanese woman who developed t-MDS/AML with t(3;21)(q26.2;q22) subsequent to receiving low-dose methotrexate (MTX) treatment for rheumatoid arthritis. Following treatment with MTX for 6 years, the patient developed anemia and neutropenia, and MTX was discontinued. A total of 3 years later, the patient was diagnosed with MDS with t(3;21)(q26.2;q22) and del (5q), which progressed rapidly to AML within 3 months. The patients was subsequently treated with azacitidine and cytarabine chemotherapy, but succumbed to the disease 6 months after diagnosis. Sequencing analysis of the nested reverse transcription-PCR products from the leukemic cells revealed the expression of two types of alternatively-spliced AME transcripts with or without RUNX1 exon 6 sequences. Western blot analysis of the leukemic cells of the patient additionally revealed that the corresponding AME fusion protein products were expressed at high levels, and that these cells also prominently expressed CCAAT/enhancer-binding protein α, the repression of which has been reported to be involved in leukemogenesis mediated by AME. To the best of our knowledge, the case discussed in the present study represents the first report of MDS/AML with t(3;21)(q26.2;q22) developing following low-dose MTX therapy for rheumatoid arthritis. Nonetheless, the clinical and molecular features of the patient in the present study were representative of those patients who typically develop this disease following exposure to chemotherapy or radiotherapy for primary malignancy, which implicates MTX in the pathogenesis of t-MDS/AML. Moreover, we confirmed the expression of two AME fusion proteins for the first time in primary leukemic cells and analyzed several cellular factors implicated in AME-mediated leukemogenesis to gain some insight into its molecular mechanisms.

CTLA4 Promotes Tyk2-STAT3-Dependent B-cell Oncogenicity.

CTL-associated antigen 4 (CTLA4) is a well-established immune checkpoint for antitumor immune responses. The protumorigenic function of CTLA4 is believed to be limited to T-cell inhibition by countering the activity of the T-cell costimulating receptor CD28. However, as we demonstrate here, there are two additional roles for CTLA4 in cancer, including via CTLA4 overexpression in diverse B-cell lymphomas and in melanoma-associated B cells. CTLA4-CD86 ligation recruited and activated the JAK family member Tyk2, resulting in STAT3 activation and expression of genes critical for cancer immunosuppression and tumor growth and survival. CTLA4 activation resulted in lymphoma cell proliferation and tumor growth, whereas silencing or antibody-blockade of CTLA4 in B-cell lymphoma tumor cells in the absence of T cells inhibits tumor growth. This inhibition was accompanied by reduction of Tyk2/STAT3 activity, tumor cell proliferation, and induction of tumor cell apoptosis. The CTLA4-Tyk2-STAT3 signal pathway was also active in tumor-associated nonmalignant B cells in mouse models of melanoma and lymphoma. Overall, our results show how CTLA4-induced immune suppression occurs primarily via an intrinsic STAT3 pathway and that CTLA4 is critical for B-cell lymphoma proliferation and survival. Cancer Res; 77(18); 5118-28. ©2017 AACR.