Recent genetic analysis has identified frequent mutations in ten-eleven translocation 2 (TET2), DNA methyltransferase 3A (DNMT3A), isocitrate dehydrogenase 2 (IDH2) and ras homolog family member A (RHOA) in nodal T-cell lymphomas, including angioimmunoblastic T-cell lymphoma and peripheral T-cell lymphoma, not otherwise specified. We examined the distribution of mutations in these subtypes of mature T-/natural killer cell neoplasms to determine their clonal architecture. Targeted sequencing was performed for 71 genes in tumor-derived DNA of 87 cases. The mutations were then analyzed in a programmed death-1 (PD1)-positive population enriched with tumor cells and CD20-positive B cells purified by laser microdissection from 19 cases. TET2 and DNMT3A mutations were identified in both the PD1+ cells and the CD20+ cells in 15/16 and 4/7 cases, respectively. All the RHOA and IDH2 mutations were confined to the PD1+ cells, indicating that some, including RHOA and IDH2 mutations, being specific events in tumor cells. Notably, we found that all NOTCH1 mutations were detected only in the CD20+ cells. In conclusion, we identified both B- as well as T-cell-specific mutations, and mutations common to both T and B cells. These findings indicate the expansion of a clone after multistep and multilineal acquisition of gene mutations.

In leukemogenesis, Notch signaling can be up and downregulated in a context-dependent manner. The transcription factor hairy and enhancer of split-1 (Hes1) is well-characterized as a downstream target of Notch signaling. Hes1 encodes a basic helix-loop-helix-type protein, and represses target gene expression. Here, we report that deletion of the Hes1 gene in mice promotes acute myeloid leukemia (AML) development induced by the MLL-AF9 fusion protein. We then found that Hes1 directly bound to the promoter region of the FMS-like tyrosine kinase 3 (FLT3) gene and downregulated the promoter activity. FLT3 was consequently upregulated in MLL-AF9-expressing immortalized and leukemia cells with a Hes1- or RBPJ-null background. MLL-AF9-expressing Hes1-null AML cells showed enhanced proliferation and ERK phosphorylation following FLT3 ligand stimulation. FLT3 inhibition efficiently abrogated proliferation of MLL-AF9-induced Hes1-null AML cells. Furthermore, an agonistic anti-Notch2 antibody induced apoptosis of MLL-AF9-induced AML cells in a Hes1-wild type but not a Hes1-null background. We also accessed two independent databases containing messenger RNA (mRNA) expression profiles and found that the expression level of FLT3 mRNA was negatively correlated with those of HES1 in patient AML samples. These observations demonstrate that Hes1 mediates tumor suppressive roles of Notch signaling in AML development, probably by downregulating FLT3 expression.

Background The transcription factor Hairy enhancer of split1 (Hes1) is well characterized as a downstream target of Notch signaling. Hes1 is a basic helix-loop-helix-type protein, and represses target gene expression. Notch signaling has been proposed to play both pro- and anti-tumorigenic roles; it promotes development of T-cell acute lymphoblastic leukemia (T-ALL), while serves as a tumor suppressor for acute myeloid leukemia (AML). Hes1 has been proven as an essential mediator of Notch signaling in T-ALL development. In contrast, we reported, in the last annual meeting, that Hes1 functions as a tumor suppressor against AML development, using a mouse model of AML induced by the MLL-AF9 fusion protein. We further explored the mechanism of Hes1-mediated suppression of AML development. Methods Common myeloid progenitors (CMPs) purified from RBP-Jf/f mouse bone marrow (BM) were serially transduced with MLL-AF9 and Cre recombinase (iCre) using retroviral vectors, and transplanted into lethally irradiated syngenic mice. CMPs from Hes1-/- mouse fetal liver were also retrovirally transduced with MLL-AF9 and transplanted after multiple rounds of replating, and then, expression levels of downstream targets were evaluated by cDNA array. Next Hes1 was retrovirally re-expressed in MLL-AF9/Hes1-/- cells and these cells were transplanted. MLL-AF9-transduced cells were treated with a hamster anti-mouse Notch agonistic antibody (Notch Ab). Results Mice transplanted with MLL-AF9/RBP-J-/- cells developed leukemia at shorter latencies than those with MLL-AF9/RBPJ+/+ cells. MLL-AF9-transduced Hes1-/- cells formed the higher number of colonies at third replating compared with MLL-AF9-transduced Hes1+/+ cells. When infused into irradiated syngenic mice, MLL-AF9/Hes1-/- cells developed leukemia at shorter latencies than MLL-AF9/ Hes1+/+ cells (MLL-AF9/Hes1-/-, 7-10 weeks, n=18 vs MLL-AF9/Hes1+/+, 10-14 weeks, n=18; p<0.001). When Hes1 was retrovirally re-expressed in MLL-AF9/Hes1-/- cells, these cells developed leukemia in recipient mice at longer latencies than mock-transduced MLL-AF9/Hes1-/- cells (Hes1/MLL-AF9, 12 weeks, n=8 vs mock/MLL-AF9, 5-7 weeks, n=7 p<0.001). When treated with an anti-Notch2 Ab, MLL-AF9/Hes1+/+ cells underwent apoptosis, whereas MLL-AF9/Hes1-/- cells did not. These results indicate that Hes1 is a definitive downstream mediator for Notch signaling-mediated suppression of AML. Among the genes with different expression levels between MLL-AF9/Hes1-/- and MLL-AF9/Hes1+/+ leukemia cells, FMS-like tyrosine kinase 3 (FLT3) was expressed at significantly higher levels in MLL-AF9/Hes1-/- leukemia cells as well as RBP-J-null Background. It was also demonstrated that FLT3 and ERK were phosphorylated with FLT3 ligand stimulation in the MLL-AF9-immortalized cells specifically with the Hes1-/- Background. An FLT3 inhibitor efficiently abrogated the proliferation of MLL-AF9/Hes1-/- leukemia cells. We accessed the independent database containing mRNA expression profiles and found that the expression level of Flt3 mRNA was negatively correlated with those of Hes1 in AML samples. Conclusion Canonical Notch signaling serves as a tumor suppressor in MLL-AF9-induced AML through upregulation of Hes1. Hes1 is an essential Notch signaling mediator for AML suppression. At least a part of Hes1 function might be explained by repression of FLT3. Disclosures: No relevant conflicts of interest to declare.

[Backgrounds] Angioimmunoblastic T-cell lymphoma (AITL) is a distinct subtype of peripheral T-cell lymphoma (PTCL), characterized by generalized lymphadenopathy and autoimmune-like manifestations. Regarding genetic lesions of AITL, frequent mutations in TET2, IDH2, DNMT3A and RHOA have been identified. In some PTCL cases, TET2 and DNMT3A mutations were identified in cell populations beyond the CD4+ T-lymphocytes, in which the tumor cells are contained, suggesting that TET2 and DNMT3A mutations occurred earlier than the commitment to CD4+ T lymphocytes. [Objective] We performed this study to identify the cell-type-specific mutations and further explore mutational profiles in AITL and AITL-related cancer. [Methods] The dataset of targeted sequencing was analyzed for 76 genes in 79 PTCL samples. Mutational origin was analyzed by cell sorting and laser microdissection. [Results] Targeted sequencing identified 168 mutations in 33 genes. Recurrent mutations, in addition to the already known frequent mutations in RHOA/TET2/IDH2/DNMT3A, were found in ODZ1 [4/79 (5%)], Notch1, NAV2, and MTERFD3 [3/79 (4%) for each], MLL2, TET3, FAT2, and LAMA2 [2/79 (3%) for each]. TET2/DMNT3A mutations showed statistically higher allelic burden than the newly identified mutations, suggesting precedence of TET2/DNMT3A mutations. Cell sorting and laser microdissection, followed by amplicon sequencing, revealed that TET2/DNMT3A mutations were identified in both tumor cell-enriched and –depleted populations while RHOA and IDH2 mutations were confined to tumor cell-enriched populations. Most of the newly identified mutations were similarly classified into the above-mentioned two types. It is noteworthy that we found some mutations only in T-cell lymphoma cell-depleted CD20-positive population but not in the tumor-cell-enriched PD-1-positive population. [Conclusion and discussion] Differentiation stages that mutational events arise are likely to be multiple in AITL and AITL-related lymphoma. Moreover, in AITL, Epstein-Bar virus-infected B cells often grow in an oligoclonal manner, sometimes resulting in monoclonal proliferation with fully malignant features. Detection of B-cell specific mutations might suggest premalignant status of B cells in these cases. Disclosures No relevant conflicts of interest to declare.