Single-cell functional genomics reveals determinants of sensitivity and resistance to natural killer cells in blood cancers.

Cancer cells can evade natural killer (NK) cell activity, thereby limiting anti-tumor immunity. To reveal genetic determinants of susceptibility to NK cell activity, we examined interacting NK cells and blood cancer cells using single-cell and genome-scale functional genomics screens. Interaction of NK and cancer cells induced distinct activation and type I interferon (IFN) states in both cell types depending on the cancer cell lineage and molecular phenotype, ranging from more sensitive myeloid to less sensitive B-lymphoid cancers. CRISPR screens in cancer cells uncovered genes regulating sensitivity and resistance to NK cell-mediated killing, including adhesion-related glycoproteins, protein fucosylation genes, and transcriptional regulators, in addition to confirming the importance of antigen presentation and death receptor signaling pathways. CRISPR screens with a single-cell transcriptomic readout provided insight into underlying mechanisms, including regulation of IFN-γ signaling in cancer cells and NK cell activation states. Our findings highlight the diversity of mechanisms influencing NK cell susceptibility across different cancers and provide a resource for NK cell-based therapies.

Venetoclax sensitivity in multiple myeloma is associated with B-cell gene expression.

Venetoclax is a highly potent, selective BCL2 inhibitor capable of inducing apoptosis in cells dependent on BCL2 for survival. Most myeloma is MCL1-dependent; however, a subset of myeloma enriched for translocation t(11;14) is codependent on BCL2 and thus sensitive to venetoclax. The biology underlying this heterogeneity remains poorly understood. We show that knockdown of cyclin D1 does not induce resistance to venetoclax, arguing against a direct role for cyclin D1 in venetoclax sensitivity. To identify other factors contributing to venetoclax response, we studied a panel of 31 myeloma cell lines and 25 patient samples tested for venetoclax sensitivity. In cell lines, we corroborated our previous observation that BIM binding to BCL2 correlates with venetoclax response and further showed that knockout of BIM results in decreased venetoclax sensitivity. RNA-sequencing analysis identified expression of B-cell genes as enriched in venetoclax-sensitive myeloma, although no single gene consistently delineated sensitive and resistant cells. However, a panel of cell surface makers correlated well with ex vivo prediction of venetoclax response in 21 patient samples and may serve as a biomarker independent of t(11;14). Assay for transposase-accessible chromatin sequencing of myeloma cell lines also identified an epigenetic program in venetoclax-sensitive cells that was more similar to B cells than that of venetoclax-resistant cells, as well as enrichment for basic leucine zipper domain-binding motifs such as BATF. Together, these data indicate that remnants of B-cell biology are associated with BCL2 dependency and point to novel biomarkers of venetoclax-sensitive myeloma independent of t(11;14).

Functional expression cloning of molecules inducing CD34 expression in bone marrow-derived stromal myofibroblasts.

We have previously shown a fraction of stromal fibroblasts/myofibroblasts (Fibs) from leukemic bone marrow cells expresses leukemia-specific transcripts along with hematopoietic and Fib-related markers. Normal bone marrow-derived Fibs (nFibs) do not express CD34 or CD45; however, nFibs may express hematopoietic markers with some specific stimulations. CD34 expression was detected in nFib cultures following the addition of a culture supernatant of blood mononuclear cells stimulated with phytohemagglutinin (PHA)-P. To identify the molecules responsible for inducing CD34 expression in nFibs, cDNA clones were isolated using functional expression cloning with a library constructed from PHA-P-stimulated human blood mononuclear cells. Positive clones inducing CD34 transcription in nFibs were selected. We confirmed that an isolated positive cDNA clone encoded human interleukin (IL)-1 beta (ß). CD34 expression was observed in the nFib cultures with recombinant human (rh) IL-1ß protein. And CD34 transcription was suppressed when a rhIL-1ß neutralizing antibody was added to the IL-1ß-stimulated nFib cultures. nFibs expressed gp130 and IL-6 receptors, and CD45 expression was detected in nFibs cultured with rhIL-1ß and rhIL-6. Chronic myelogenous leukemia (CML) cells reportedly respond well to IL-1ß. When CML-derived Fibs were cultured with rhIL-1ß and rhIL-6, CD45-positive cells increased in number. Cell fate may be influenced by an external specific stimulation without gene introduction.

Highly specific intracellular ubiquitination of a small molecule.

Ubiquitin is a small, highly conserved protein that acts as a posttranslational modification in eukaryotes. Ubiquitination of proteins frequently serves as a degradation signal, marking them for disposal by the proteasome. Here, we report a novel small molecule from a diversity-oriented synthesis library, BRD1732, that is directly ubiquitinated in cells, resulting in dramatic accumulation of inactive ubiquitin monomers and polyubiquitin chains causing broad inhibition of the ubiquitin-proteasome system. Ubiquitination of BRD1732 and its associated cytotoxicity are stereospecific and dependent upon two homologous E3 ubiquitin ligases, RNF19A and RNF19B. Our finding opens the possibility for indirect ubiquitination of a target through a ubiquitinated bifunctional small molecule, and more broadly raises the potential for posttranslational modification in trans.

Type 1 diabetes mellitus after cord blood transplantation from an unrelated donor with a disease-sensitive haplotype.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT), a curative treatment for hematopoietic neoplasms, often causes various autoimmune disease-like conditions. In contrast, allo-HSCT-related type 1 diabetes mellitus is extremely rare. Herein, we report a case of allo-HSCT-related type 1 diabetes mellitus in a patient who had undergone cord blood transplantation (CBT) as a treatment for acute myeloid leukemia. The patient's human leukocyte antigen was replaced with the donor type after transplantation. The donor had a disease-sensitive haplotype. To the best of our knowledge, this is the first reported case of type 1 diabetes mellitus following CBT.

Genome-scale functional genomics identify genes preferentially essential for multiple myeloma cells compared to other neoplasias.

Clinical progress in multiple myeloma (MM), an incurable plasma cell (PC) neoplasia, has been driven by therapies that have limited applications beyond MM/PC neoplasias and do not target specific oncogenic mutations in MM. Instead, these agents target pathways critical for PC biology yet largely dispensable for malignant or normal cells of most other lineages. Here we systematically characterized the lineage-preferential molecular dependencies of MM through genome-scale clustered regularly interspaced short palindromic repeats (CRISPR) studies in 19 MM versus hundreds of non-MM lines and identified 116 genes whose disruption more significantly affects MM cell fitness compared with other malignancies. These genes, some known, others not previously linked to MM, encode transcription factors, chromatin modifiers, endoplasmic reticulum components, metabolic regulators or signaling molecules. Most of these genes are not among the top amplified, overexpressed or mutated in MM. Functional genomics approaches thus define new therapeutic targets in MM not readily identifiable by standard genomic, transcriptional or epigenetic profiling analyses.

Etanercept for the treatment of intractable hemophagocytic syndrome with systemic lupus erythematosus.

A 41-year-old woman presented with continuous fever, and her laboratory data suggested the recrudescence of systemic lupus erythematosus. She was treated with 60 mg/day prednisolone. With a dose reduction of prednisolone, high fever and pancytopenia were observed again. A bone marrow biopsy revealed hemophagocytosis. The effects of steroid pulse therapy, high-dose intravenous immunoglobulin, cyclosporine A, and methotrexate were insufficient. However, after four injections of etanercept (25 mg, twice a week) subcutaneously, her symptoms had completely resolved. In such cases, therapy with etanercept may be effective.

The FLT3 internal tandem duplication mutation at disease diagnosis is a negative prognostic factor in myelodysplastic syndrome patients.

The role of the FMS-like tyrosine kinase 3 (FLT3) gene in acute myeloid leukemia (AML) has been well documented and the FLT3-internal tandem duplication (FLT3-ITD) mutation has been identified as a prognostic factor in AML. Due to its low incidence, the role of the FLT3 mutation remains unclear in myelodysplastic syndrome (MDS) patients. To investigate the impact of the FLT3-ITD status on the prognosis of MDS at diagnosis, we retrospectively analyzed 72 MDS patients admitted to Teikyo University Hospital. FLT3-ITD was examined by a reverse transcription-polymerase chain reaction using complementary DNA synthesized from mRNA extracted from bone marrow mononuclear cells at the diagnosis of MDS. Fifteen patients (20.8 %) were positive for FLT3-ITD and had significantly worse overall survival (OS) and progression-free survival (PFS) than patients who were negative (P < 0.001 and P < 0.001, respectively) in a multivariate analysis. We also investigated whether the Wilms' tumor gene-1 (WT-1) copy number was associated with the FLT3-ITD mutational status using data available on WT-1 from 57 patients. A WT-1 transcript copy number ≥50/µg total mRNA in peripheral blood was detected in 35 patients (61.4 %). All FLT3-ITD-positive patients showed WT-1 ≥50. The FLT3-ITD-positive group showed significantly higher WT-1 transcription levels than the negative group. These results indicate that the FLT3-ITD mutation has a prognostic impact at the diagnosis of MDS and is associated with a high level of WT-1.

Serum high-density lipoprotein cholesterol level has a significant prognostic impact on outcomes of follicular lymphoma patients.

We investigated the potential of nutritional and inflammatory parameters as prognostic factors for follicular lymphoma (FL), and also examined the predictive value of the early progression of disease within 24 months of first-line chemo-immunotherapy (POD24). We retrospectively analyzed 46 patients with FL admitted to Teikyo University Hospital and treated with chemo-immunotherapy between May 2009 and July 2019. Physical characteristics, blood parameters, and markers or scores for consumptive/inflammatory and nutritional conditions were used as variables. Nine parameters correlated with poor overall survival (OS) in univariate analysis: An Eastern Cooperative Oncology Group (ECOG) scale performance status (PS) ≥2, five or more involved nodal sites, positive bone marrow (BM) involvement, a serum albumin level <3.5 g/dL, CRP >0.5 mg/dL, lactate dehydrogenase (LD) higher than the upper normal limit (UNL), high-density lipoprotein cholesterol (HDL-C) <40 mg/dL, modified Glasgow prognostic score of 1-2, and the geriatric nutritional risk index <82. In multivariate analysis, ECOG PS ≥2, positive BM involvement, and a serum HDL-C level <40 mg/dL remained significant for poor progression-free survival. One-year OS rate after receiving salvage chemotherapy was lower in the POD24 group (50%) and POD24 correlated with ECOG PS ≥2, positive BM involvement, a serum lactate dehydrogenase >UNL, and HDL-C <40 mg/dL by Fisher's exact test. These results indicate that low serum HDL-C levels appear to be important for predicting the risk of POD24 and the worse prognosis of FL.

[Acute myelogenous leukemia developed at the 26th week of gestation].

We report here a 35-year-old pregnant woman with acute myelogenous leukemia (AML). She was diagnosed with AML (M2) in August 2009, coinciding with the 26(th) week of pregnancy. She underwent a cesarean section at 27 weeks gestation, delivering a very low birth weight male infant (1,066 g). One week later, she received induction chemotherapy with idarubicin and cytarabine. She achieved complete remission after two courses of chemotherapy. The incidence of acute leukemia during pregnancy is low. Chemotherapy after the 2(nd) trimester is not associated with an increased rate of fetal malformation. However, there are some reports that in utero exposure to chemotherapy during any trimester of pregnancy carries a significant risk for an unfavorable outcome including low birth weight, fetal or neonatal death, and intrauterine growth retardation. Decision on the choice of treatment for acute leukemia during pregnancy should be case-dependent. If an infant has grown sufficiently to be viable outside uterus and the patient does not demonstrate a severe bleeding tendency, delivery by cesarean section preceding chemotherapy is one option.

An Embryonic Diapause-like Adaptation with Suppressed Myc Activity Enables Tumor Treatment Persistence.

Treatment-persistent residual tumors impede curative cancer therapy. To understand this cancer cell state we generated models of treatment persistence that simulate the residual tumors. We observe that treatment-persistent tumor cells in organoids, xenografts, and cancer patients adopt a distinct and reversible transcriptional program resembling that of embryonic diapause, a dormant stage of suspended development triggered by stress and associated with suppressed Myc activity and overall biosynthesis. In cancer cells, depleting Myc or inhibiting Brd4, a Myc transcriptional co-activator, attenuates drug cytotoxicity through a dormant diapause-like adaptation with reduced apoptotic priming. Conversely, inducible Myc upregulation enhances acute chemotherapeutic activity. Maintaining residual cells in dormancy after chemotherapy by inhibiting Myc activity or interfering with the diapause-like adaptation by inhibiting cyclin-dependent kinase 9 represent potential therapeutic strategies against chemotherapy-persistent tumor cells. Our study demonstrates that cancer co-opts a mechanism similar to diapause with adaptive inactivation of Myc to persist during treatment.

Functional Genomics Identify Distinct and Overlapping Genes Mediating Resistance to Different Classes of Heterobifunctional Degraders of Oncoproteins.

Heterobifunctional proteolysis-targeting chimeric compounds leverage the activity of E3 ligases to induce degradation of target oncoproteins and exhibit potent preclinical antitumor activity. To dissect the mechanisms regulating tumor cell sensitivity to different classes of pharmacological "degraders" of oncoproteins, we performed genome-scale CRISPR-Cas9-based gene editing studies. We observed that myeloma cell resistance to degraders of different targets (BET bromodomain proteins, CDK9) and operating through CRBN (degronimids) or VHL is primarily mediated by prevention of, rather than adaptation to, breakdown of the target oncoprotein; and this involves loss of function of the cognate E3 ligase or interactors/regulators of the respective cullin-RING ligase (CRL) complex. The substantial gene-level differences for resistance mechanisms to CRBN- versus VHL-based degraders explains mechanistically the lack of cross-resistance with sequential administration of these two degrader classes. Development of degraders leveraging more diverse E3 ligases/CRLs may facilitate sequential/alternating versus combined uses of these agents toward potentially delaying or preventing resistance.

Acute myelogenous leukemia cells with the MLL-ELL translocation convert morphologically and functionally into adherent myofibroblasts.

Bone marrow-myofibroblasts, a major component of bone marrow-stroma, are reported to originate from hematopoietic stem cells. We show in this paper that non-adherent leukemia blasts can change into myofibroblasts. When myeloblasts from two cases of acute myelogenous leukemia with a fusion product comprising mixed lineage leukemia and RNA polymerase II elongation factor, were cultured long term, their morphology changed to that of myofibroblasts with similar molecular characteristics to the parental myeloblasts. The original leukemia blasts, when cultured on the leukemia blast-derived myofibroblasts, grew extensively. Leukemia blasts can create their own microenvironment for proliferation.

Direct Factor X sequestration by systemic amyloid light-chain amyloidosis.

We present a lymphoplasmacytic lymphoma patient with Factor X (FX) deficiency. Despite the absence of FX inhibitor, the administration of fresh frozen plasma and anti-inhibitor coagulant complex did not increase the FX level. The autopsy showed that massive amyloid depositions to multiple organs and FX existed in union with amyloidosis.

Molecular analysis of chronic eosinophilic leukemia with t(4;10) showing good response to imatinib mesylate.

A 38-year-old Japanese man was referred to our hospital in June 2003 for treatment of acute respiratory failure with severe eosinophilia. Idiopathic hypereosinophilic syndrome had been diagnosed in 1994. However, karyotypic examination of bone marrow cells revealed that chromosomal translocation with t(4;10)(q12;p11) had occurred in 2000, and chronic eosinophilic leukemia was diagnosed. At admission, the patient's respiratory condition was extremely serious, and mechanical support was necessary. Despite treatment with steroid pulse therapy and cytarabine, the blood eosinophil count did not decrease, and the patient's respiratory condition worsened. After obtaining informed consent, we administered imatinib mesylate at a dose of 200 mg/day for 2 days and 100 mg/day for 3 days. The blood eosinophil count decreased dramatically over 5 days, and the patient's condition rapidly improved, such that the patient could be discharged. In this case, we performed molecular analysis using peripheral blood. The FIP1-like 1 (FIP1L1)-platelet-derived growth factor receptor alpha (PDGFRalpha) fusion transcript was found with the reverse transcriptase polymerase chain reaction analysis. In this case, eosinophilia was possibly caused by constitutive activation of tyrosine kinase produced by the FIP1L1-PDGFRalpha fusion transcript.

Colonic EBV-Associated Lymphoproliferative Disorder in a Patient Treated with Rabbit Antithymocyte Globulin for Aplastic Anemia.

Epstein-Barr-virus- (EBV-) associated lymphoproliferative disorder (LPD) after immunosuppressive therapy for aplastic anemia (AA), in a nontransplant setting, has not been well described. We report one case of colonic EBV-LPD after a single course of immunosuppressive therapy for AA. The patient developed multiple colonic tumors 3 months after receiving immunosuppressive therapy, which consisted of rabbit antithymocyte globulin (ATG), cyclosporine, and methyl-predonisolone. The histological findings of biopsy specimens revealed that atypical lymphocytes had infiltrated colonic glands. Immunohistochemical staining for CD20 was positive, and in situ hybridization for EBV-encoded small RNAs was also positive. The EBV viral load in peripheral blood was slightly increased to 140/10(6) white blood cells. After the cessation of immunosuppressant, the colonic tumors spontaneously regressed, and the EBV viral load decreased to undetectable levels. This is the first report of the single use of rabbit ATG inducing colonic EBV-LPD. Because a single use of immunosuppressive therapy containing rabbit ATG can cause EBV-LPD, we should carefully observe patients receiving rabbit ATG for AA.

Chronic Myelogenous Leukemia Cells Contribute to the Stromal Myofibroblasts in Leukemic NOD/SCID Mouse In Vivo.

We recently reported that chronic myelogenous leukemia (CML) cells converted into myofibroblasts to create a microenvironment for proliferation of CML cells in vitro. To analyze a biological contribution of CML-derived myofibroblasts in vivo, we observed the characters of leukemic nonobese diabetes/severe combined immunodeficiency (NOD/SCID) mouse. Bone marrow nonadherent mononuclear cells as well as human CD45-positive cells obtained from CML patients were injected to the irradiated NOD/SCID mice. When the chimeric BCR-ABL transcript was demonstrated in blood, human CML cells were detected in NOD/SCID murine bone marrow. And CML-derived myofibroblasts composed with the bone marrow-stroma, which produced significant amounts of human vascular endothelial growth factor A. When the parental CML cells were cultured with myofibroblasts separated from CML cell-engrafted NOD/SCID murine bone marrow, CML cells proliferated significantly. These observations indicate that CML cells make an adequate microenvironment for their own proliferation in vivo.