Although treatment advances over recent decades have significantly improved survival of patients with multiple myeloma, there is still an unmet medical need for more effective treatments. In this study, we identified G-protein-coupled receptor family C group 5 member D (GPRC5D) expression on the surface of malignant cells involved in multiple myeloma, but except for plasma cells and B cells, not at appreciable levels on normal hematopoietic cells and bone marrow progenitors , including hematopoietic stem cells. In addition, we constructed IgG-based anti-GPRC5D/CD3 bispecific T-cell-redirecting antibodies (GPRC5D TRAB), which suppressed the tumor growth of GPRC5D-positive myeloma cells through the activation of T cells in vitro and in vivo in xenograft models. Collectively, these findings suggest that GPRC5D is an antigen specific to multiple myeloma and a potential target of TRAB therapy.
Acoustic plane focussing and a scanning mirror enhances throughput of an imaging cytometer.
A 64-year-old woman presented with generalized lymphadenopathy and systemic manifestations. The examination of a biopsy specimen revealed peripheral T-cell lymphoma not otherwise specified (PTCL-NOS) expressing cytotoxic molecules. Umbilical cord blood transplantation was successful during a partial remission state after the administration of salvage chemotherapy. The donor-derived large granular lymphocytes started to increase as a result of cytomegalovirus reactivation. The fraction of natural killer (NK) cells expressing the NKG2C molecule accounted for one-third of the total lymphocytes for almost two years. We implicitly indicate the association between the persistence of donor-derived NKG2C+ NK cell-expansion and maintaining a complete remission in similar cases of aggressive PTCL-NOS.
Cancer-specific metabolic activities play a crucial role in the pathogenesis of human malignancies. To investigate human acute leukemia-specific metabolic properties, we comprehensively measured the cellular metabolites within the CD34+ fraction of normal hematopoietic stem progenitor cells (HSPCs), and primary human acute myelogenous leukemia (AML) and lymphoblastic leukemia (ALL) cells. Here we show that human leukemia addicts to the branched-chain amino acid (BCAA) metabolism to maintain their stemness, irrespective of myeloid or lymphoid types. Human primary acute leukemias had BCAA transporters for BCAA uptake, cellular BCAA, α-ketoglutarate (α-KG) and cytoplasmic BCAA transaminase-1 (BCAT1) at significantly higher levels than control HSPCs. Isotope-tracing experiments showed that in primary leukemia cells, BCAT1 actively catabolizes BCAA using α-KG into branched-chain α-ketoacids (BCKAs), whose metabolic processes provide leukemia cells with critical substrates for the TCA cycle and the non-essential amino acids synthesis, both of which reproduce α-KG to maintain its cellular level. In xenogeneic transplantation experiments, deprivation of BCAA from daily diet strongly inhibited expansion, engraftment and self-renewal of human acute leukemia cells. Inhibition of BCAA catabolism in primary AML or ALL cells specifically inactivates polycomb repressive complex 2 (PRC2) function, an epigenetic regulator for stem cell signatures, through inhibiting transcription of PRC components, such as zeste homolog 2 (EZH2) and embryonic ectoderm development (EED). Accordingly, BCAA catabolism plays an important role in maintenance of stemness in primary human AML and ALL, and molecules related to the BCAA metabolism pathway should be critical targets for acute leukemia treatment.
Alternative polyadenylation (APA) can alter the three prime untranslated region (3'UTR) length of mRNAs, crucial for regulating mRNA metabolism and gene expression. Despite the prevalence of APA post-transcriptional regulation in cancers, changes in 3'UTR length by APA and its contribution to leukemia development have not been thoroughly studied. In this study, we demonstrated the significance of APA of leukemic fusion genes in acute myelogenous leukemia (AML) development. T(8;21) is the most common chromosomal abnormality in AML and encodes the AML1-ETO (AE) fusion gene. The AE 3'UTR has a full length of 5.2kb that contains 4 canonical polyadenylation sites (PAS), such that APA can result in several mRNA isoforms with varying 3'UTR lengths. However, RNA-seq and absolute quantification qPCR revealed that AE mainly uses 2 PAS: 5.2kb (long 3'UTR) and 3.7kb (short 3'UTR). Moreover, the AE short 3'UTR is the major isoform in both t(8;21) AML primary patients and cell lines. Based on these findings, we next hypothesize that changes in PAS usage and thus AE 3'UTR length, can modulate fusion gene expression. The single cell based dynamic array revealed that AE+ non-leukemic differentiated cells coexisted with AML blasts in the diagnostic bone marrow and these non-leukemic AE+ cells expressed much lower AE compared to AML blasts (monocytes, 0.19-fold and granulocytes, 0.11-fold, p<0.001). Among these leukemic and AE+ non-leukemic cells, AE expression and long 3'UTR usage showed a significant negative correlation (R2=0.64, p<0.01). Moreover, half-life assay using actinomycin D showed that AE long 3'UTR had much shorter half-life than AE short 3'UTR (0.8 hour and 2.8 hours, p<0.01). A dual reporter assay also presented the predominant repression of AE long 3'UTR (3.7 kb: 0.31 and 5.2 kb: 0.08, p<0.001). These data suggest that AE 3'UTR length affects mRNA stability and is an important indicator of fusion gene expression. The selective inhibition of AE short 3'UTR PAS by morpholino antisense oligonucleotide caused an increase of AE long 3'UTR usage, decrease of total AE expression, and an increase in apoptosis. These results indicate that the inhibition of AE short 3'UTR PAS increases the long 3'UTR usage and the PAS usage of leukemic fusion genes could be a potential therapeutic target. To find the key regulator of this 3'UTR change, we comprehensively knocked down all APA machinery members by shRNA in t(8;21) AML cell lines. We discovered that CPSF1 (cleavage and polyadenylation specific factor 1) knockdown increased long 3'UTR usage (37% in total AE) and down-regulated overall AE mRNA (0.47-fold). Importantly, CPSF1 was highly expressed in t(8;21) AML patients' leukemia cells compared to healthy CD34+ cells (2.7-fold, p=0.01). CPSF1 knockdown also promoted apoptosis and Gene Set Enrichment Analysis revealed that CPSF1 knockdown showed the similar gene signature to AE knockdown. These data suggest that CPSF1 promotes AE 3'UTR shortening, resulting in AE stability and a growth advantage of AML cells. Importantly, we also found that AE regulates CPSF1 expression, suggesting a positive feedback loop of AML1-ETO and CPSF1 in t(8;21) AML. Finally, we also checked APA of leukemic fusion gene MLL-AF9, which has 2 canonical PAS (0.1kb short 3'UTR and 0.7kb long 3'UTR). Compared to healthy CD34+ cells, THP-1 cells (MLL-AF9+ AML cell line) predominantly used MLL-AF9 short 3'UTR. Knockdown of CPSF1, which is also up-regulated in MLL-associated AML, showed increase of MLL-AF9 long 3'UTR usage, decrease of MLL-AF9 expression, and an increase of apoptosis in THP-1 cells. Collectively, we conclude that the polyadenylation regulator CPSF1 controls 3'UTR length of leukemic fusion transcripts, and extending the fusion gene 3'UTR by knocking down CPSF1 reduces fusion gene mRNA stability and restricts cell growth in both AE+ and MLL-AF9+ AMLs. These results suggest that 3'UTR shortening of oncogenic fusion gene transcripts contributes to leukemogenesis and controlling CPSF1 and 3'UTR usage may be a useful approach to inhibit leukemia progression. Disclosures Akashi: Pfizer: Research Funding; Taiho Pharmaceutical: Research Funding; Kyowa Hakko Kirin: Research Funding, Speakers Bureau; Bristol-Myers Squibb: Research Funding, Speakers Bureau; Eli Lilly Japan: Research Funding; Otsuka Pharmaceutical: Research Funding; Eisai: Research Funding; Astellas Pharma: Research Funding; MSD: Research Funding; sanofi: Research Funding; Novartis pharma: Research Funding; Chugai Pharma: Research Funding; Celgene: Research Funding, Speakers Bureau; Asahi-kasei: Research Funding; Ono Pharmaceutical: Research Funding.
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