ALBERT

Description: Nowadays, patients with multiple myeloma (MM) have multiple choices of therapy including monoclonal antibodies, proteasome inhibitors, and immunomodulatory drugs (IMiDs), whereas some patients still develop resistance to these drugs and require novel therapeutic modalities. Here, we focused on inhibition of HDAC and AKT to overcome drug resistance. Lenalidomide (Len) selectively binds to cereblon (CRBN), which mediates recruitment of specific substrates like IKZF1 to E3 ubiquitin ligase and subsequent degradation, resulting in downregulation of IRF-4 and c-Myc. Then, we developed Len-resistant myeloma cells by RNAi-mediated downregulation of CRBN. Treatment of these cells with HDAC inhibitors reduced IKZF1 mRNA, suggesting potential efficacy of HDAC inhibitors against CRBN-low expressing or mutated MM. According to the integrated database for expression profile and disease prognosis (GenomicScape, http://www.genomicscape.com), higher expression of MICA was significantly associated with better overall survival in MM. MICA is an NK cell-activating ligand and plays an important role in ADCC. We observed that ADCC activity of both daratumumab and elotuzumab against MM cells was enhanced in the presence of HDAC inhibitors, which was compatible with our previous data that HDAC inhibitors upregulated MICA mRNA expression via inhibition of IKZF1 (ASH2018 abstract #4435). We also observed that HDAC inhibitors upregulated MICA mRNA in CRBN-deficient cells, suggesting promise of the combination of HDAC inhibitors and monoclonal antibodies against Len-resistant MM. Len-resistance is also affected by phosphorylation status of GSK-3. PI3K/AKT pathway is frequently activated in MM cells, and AKT inactivates GSK-3 by direct phosphorylation, resulting in c-Myc stabilization. Enhanced phosphorylation of GSK-3 was observed in CRBN-deficient H929 cells after long-term culture with Len, and such a phosphorylation status of GSK-3 was correlated with less CRBN amount and higher Len concentration (Figure 1). Afuresertib, an AKT inhibitor, suppressed GSK-3 phosphorylation (p-GSK-3) with or without ACY-1215, an HDAC inhibitor, leading to a substantial decrease of c-Myc (Figure 2). On the other hand, CHIR 99021, a GSK-3 inhibitor, partially counteracted to cytotoxic effect of afuresertib on H929 cells (Figure 3). These results suggest that increased p-GSK-3 is involved in acquired Len-resistance, and that combined inhibition of HDAC and AKT can overcome Len-resistance through decreased p-GSK-3. Furthermore, we examined the efficacy of CUDC-907, a dual HDAC and PI3K inhibitor. CUDC-907 had a cytotoxic effect on the MM cell lines including those had low CRBN expression. Bortezomib, doxorubicin, and dexamethasone resistant MM cell lines were also sensitive to CUDC-907. CUDC-907 upregulated MICA mRNA expression, but downregulated IKZF1 mRNA expression. Treatment of RPMI-8226 cells with CUDC-907 enhanced the ADCC activity of daratumumab (Figure 4). Furthermore, CUDC-907 was effective on primary MM cells which were resistant to bortezomib and Len (Figure 5). Thus, dual inhibition of HDAC and AKT with or without monoclonal antibodies is a promising therapeutic approach to multi-drug resistant MM. Disclosures Imai: Celgene: Honoraria, Research Funding; Janssen Parmaceutical K.K.: Honoraria, Research Funding; Bristol-Myers Squibb: Research Funding. Futami:Torii Pharmaceutical: Research Funding. Ri:Janssen Pharmaceutical: Honoraria, Research Funding; Takeda Pharmaceutical: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Daiichi Sankyo: Research Funding; Ono Pharmaceutical: Honoraria, Research Funding; Kyowa Kirin: Research Funding; Chugai Pharmaceutical: Research Funding; Sanofi: Honoraria, Research Funding; Abbvie: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; MSD: Research Funding; Novartis Pharma: Research Funding; Gilead Sciences: Research Funding; Astellas Pharma: Research Funding; Teijin Pharma: Research Funding. Yasui:TokioTHERA Holdings, Inc.: Equity Ownership. Iida:Teijin Pharma: Research Funding; Celgene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Takeda: Honoraria, Research Funding; Astellas: Research Funding; Gilead: Research Funding; Sanofi: Research Funding; MSD: Research Funding; Abbvie: Research Funding; Kyowa Kirin: Research Funding; Chugai: Research Funding; Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Daichi Sankyo: Honoraria, Research Funding. Tojo:Torii Pharmaceutical: Research Funding; AMED: Research Funding.

Description: AML1, a gene on chromosome 21 encoding a transcription factor, is disrupted in the (8;21)(q22;q22) and (3;21)(q26;q22) chromosomal translocations associated with myelogenous leukemias; as a result, chimeric proteins AML1/ETO(MTG8) and AML1/Evi-1 are generated, respectively. To clarify the roles of AML1/ETO(MTG8) and AML1/Evi-1 in leukemogenesis, we investigated subcellular localization of these chimeric proteins by immunofluorescence labeling and subcellular fractionation of COS-7 cells that express these chimeric proteins. AML1/ETO(MTG8) and AML1/Evi-1 are nuclear proteins, as is wild-type AML1. Polyomavirus enhancer binding protein (PEBP)2β(core binding factor [CBF]β), a heterodimerizing partner of AML1 that is located mainly in the cytoplasm, was translocated into the nucleus with dependence on the runt domain of AML1/ETO(MTG8) or AML1/Evi-1 when coexpressed with these chimeric proteins. When a comparable amount of wild-type AML1 or the chimeric proteins was coexpressed with PEBP2β(CBFβ), more of the cells expressing the chimeric proteins showed the nuclear accumulation of PEBP2β(CBFβ), as compared with the cells expressing wild-type AML1. We also showed that the chimeric proteins associate with PEBP2β(CBFβ) more effectively than wild-type AML1. These data suggest that the chimeric proteins are able to accumulate PEBP2β(CBFβ) in the nucleus more efficiently than wild-type AML1, probably because of the higher affinities of the chimeric proteins for PEBP2β(CBFβ) than that of wild-type AML1. These effects of the chimeric proteins on the cellular distribution of PEBP2β(CBFβ) possibly cause the dominant negative properties of the chimeric proteins over wild-type AML1 and account for one of the mechanisms through which these chimeric proteins contribute to leukemogenesis.

Description: Cancer immunotherapy using chimeric antigen receptor-armed T cells (CAR-T cells) have shown excellent outcomes in hematological malignancies. However, cytokine release syndrome (CRS), characterized by excessive activation of CAR-T cells and macrophages remains to be overcome. Steroid administration usually resolves signs and symptoms of CRS but abrogates CAR-T cell expansion and persistence. Tocilizumab, a humanized monoclonal antibody against interleukin-6 receptor (IL-6R), attenuates CRS without significant loss of CAR-T cell activities, while perfect rescue of CRS symptoms cannot be achieved by IL-6/IL-6R blockade. There is actual need for novel strategies to prevent or cure CRS. TO-207, an N-benzoyl-L-phenylalanine derivative compound, significantly inhibits inflammatory cytokine production in a human monocyte/macrophage-specific manner. Here we tested TO-207 for its ability to inhibit cytokine production without impaired CAR-T cell function in a CRS-simulating co-culture system consisting of CAR-T cells, target leukemic cells and monocytes. To observe a precise pattern of cytokine release from CAR-T cells and monocytes, we first established a co-culture system that mimics CRS using K562/CD19 cells, 19-28z CAR-T cells, and peripheral blood CD14+ cells. IFN-γ was produced exclusively from CAR-T cells, and TNF-α, MIP-1α, M-CSF, and IL-6 were produced from both CAR-T cells and monocytes, but monocytes were the major source of these cytokine production. MCP-1, IL-1β, IL-8, and IL-10 were released exclusively from monocytes. To observe the effect of drugs on cytokine production, prednisolone (PSL), TO-207, tocilizumab, and anakinra (an IL-1R antagonist) were added to the co-culture. PSL exhibited suppressive effects on TNF-α and MCP-1 production. Tocilizumab did not suppress these cytokines. Anakinra up-regulated IL-6 and IL-1β production, probably due to activation of negative feedback loops. Interestingly, TO-207 widely suppressed all of these monocyte-derived cytokines including TNF-α, IL-6, IL-1β, MCP-1, IL-8, and GM-CSF. Next, we observed whether the cytokine inhibition by TO-207 attenuates killing effect of CAR-T cells. PSL attenuated killing effect of CD4+ CAR-T cells and CD8+ CAR-T cells toward K562/CD19 cells. In contrast, TO-207 did not exhibit any change in cytotoxicity of CD4+ CAR-T cells and CD8+ CAR-T cells. To determine whether the effect of PSL and TO-207 on cytotoxicity changes in the presence of CD14+ monocytes, CD14+ cells were added to the co-culture. In the absence of CAR-T cells, PSL induced a modest attenuation of cytotoxicity, whereas to the CAR-T cells, PSL exhibited a significant attenuation of cytotoxicity. TO-207 exhibited a minimal effect on cytotoxicity in the absence or presence of CAR-T cells. These results suggested that CAR-T cells play a major role in the cytotoxicity toward leukemia cells, and drugs that do not affect CAR-T cell functions, such as TO-207, maintain their cytotoxic effects on leukemia cells. In conclusion, our present co-culture model with K562/CD19 cells, 19-28z CAR-T cells, and CD14+ monocytes accurately recapitulate killing effect and cytokine release profiles. IFN-γ was produced exclusively by CAR-T cells, but majority of other cytokines such as TNF-α, MIP-1α, M-CSF, IL-6, MCP-1, IL-1β, IL-8, and IL-10 were from CD14+ monocytes/macrophages. Because killing effect was largely dependent on CAR-T cells while cytokine production was dependent on monocytes/macrophages, selective inhibition of pro-inflammatory cytokines from monocytes by TO-207 would be ideal for treatment of CAR-T-related CRS. These results encourage us to consider a clinical application for CRS. Figure Disclosures Futami: Torii Pharmaceutical: Research Funding. Suzuki:Torii Pharmaceutical: Employment. Kato:Torii Pharmmaceutical: Research Funding. Tahara:Torii Pharmaceutical: Employment. Imai:Celgene: Honoraria, Research Funding; Janssen Pharmaceutical K.K: Honoraria, Research Funding; Bristol-Myers Squibb: Research Funding. Mimura:Torii Pharmaceutical: Employment. Watanabe:Torii Pharmaceutical: Employment. Tojo:AMED: Research Funding; Torii Pharmaceutical: Research Funding.

Description: The AML1 gene encodes a DNA-binding protein that contains the runt domain and is the most frequent target of translocations associated with human leukemias. Here, point mutations of the AML1 gene, V105ter (single-letter amino acid code) and R139G, (single-letter amino acid codes) were identified in 2 cases of myelodysplastic syndrome (MDS) by means of the reverse transcriptase–polymerase chain reaction single-strand conformation polymorphism method. Both mutations are present in the region encoding the runt domain of AML1 and cause loss of the DNA-binding ability of the resultant products. Of these mutants, V105ter has also lost the ability to heterodimerize with polyomavirus enhancer binding protein 2/core binding factor β (PEBP2β/CBFβ). On the other hand, the R139G mutant acts as a dominant negative inhibitor by competing with wild-type AML1 for interaction with PEBP2β/CBFβ. This study is the first report that describes mutations of AML1 in patients with MDS and the mechanism whereby the mutant acts as a dominant negative inhibitor of wild-type AML1.

Description: Background: Langerhans cell histiocytosis (LCH) is a rare inflammatory myeloid neoplasm characterized by aberrant expansion of CD1a+CD207+ Langerhans-like cells with a variable infiltration of immune cells. Although the precise pathophysiology is to be elucidated, a frequent occurrence of oncogenic BRAF-V600E mutation in high-risk patients (Badalian-Very, et al. Blood. 2010) has made it one of the candidates of major driver mutations in LCH as well as in hairy cell leukemia. Its onset in adulthood is rather infrequent as compared with that in childhood, and the clinical profile and treatment outcome of adult LCH is poorly documented. Here, we report the clinical features and BRAF status of Japanese patients with adult LCH in a single institution. Methods: Between 2005 and 2016, 30 adult LCH patients with histopathological diagnosis and/or characteristic radiological findings were referred to our hospital for evaluation and treatment. In almost all cases, we re-evaluated the involved organs and determined the clinical subtypes. For detection of BRAF-V600E, cell-free DNA (cfDNA) was prepared from plasma of patients and was subjected to genotyping of BRAF mutations by allele-specific Q-PCR, which was specifically designed for detection of BRAF-V600E with a 3'-phosphate-modified oligonucleotide blocker as previously described (Kobayashi M and Tojo A. Blood. 2014). Mutant BRAF load was estimated from the standard curve in each assay and was expressed as the percentage of mutant alleles to total number of alleles. Considering the safety and efficacy at outpatient clinic without hospitalization, we adopted 9 cycles of JLSG Special C regimen as a first-line therapy for the patients who require treatment. The protocol is consisted of 6mg vinblastine (VBL) on Day1, 60mg oral prednisolone (PSL) on Day1-5, and 20mg/m2 methotrexate (MTX) on Day15 with 1.5mg/kg daily 6-mercaptopurine (6MP) (Morimoto A, et al. Int J Hematol. 2013). Results: Patients' median age at diagnosis was 43 (range 24-66) and 50% was female. Fourteen patients (47%) had a single organ disease (single-system; Ss) and others had multi-organ disease (multi-system; Ms). Bone (50%) and skin (29%) were main lesions in Ss patients, who were younger (median 39) than Ms patients (median 44). Nine patients (30%) developed diabetes insipidus caused by pituitary lesions. Pulmonary involvement was found in 6 patients (20%), and all except one were heavy smokers at the onset or relapse of LCH in these patients. Among Ms patients, there were 8 high-risk patients with lung, liver, spleen or bone marrow involvement. We performed ASQ-PCR analysis of BRAF mutation in 20 patients (12 Ms patients and 8 Ss patients), and only 4 Ms patients were positive for V600E mutant. Twenty-three patients received Special C regimen and 4 refractory patients were followed by 2-chlorodeoxyadenosine (2-CdA) as a salvage therapy. After Special C regimen with or without a salvage therapy, five patients had relapsed. Three of them received once more Special C regimen and two among them showed improvement. During their clinical courses, only two Ms patients, who were positive for BRAF-V600E and received 2-CdA, died of LCH progression and a cerebrovascular event, respectively. Conclusion: Since adult LCH is quite rare and most likely ignored by physicians, the diagnosis and the clinical intervention is generally delayed. Nonetheless, most of patients benefited from the low-intensity chemotherapy and their prognosis is rather favorable. Recent findings of MAPK and PI3K pathway mutation in LCH will facilitate the understanding of its pathogenesis and development of its novel molecular therapy. Disclosures No relevant conflicts of interest to declare.

Description: Aberrant oligomerization of transcription factors has recently emerged as a prevalent mechanism for activating their oncogenic potential in hematopoietic malignancies. Furthermore, for some chimeric transcription factors generated in leukemia-associated chromosomal translocations including PML-RARα and AML1-ETO, it has also been proposed that aberrant recruitment of corepressor complexes caused by oligomerization is critical for the development of leukemia. Among leukemia-associated transcription factors is Evi-1, which is frequently activated in cases with myeloid malignancies. Evi-1a and Evi-1c, with the latter also called MDS1-Evi-1, are two alternative forms derived from the Evi-1 gene. Evi-1a is a Zn finger transcription factor which recruits CtBP as a transcriptional corepressor and possesses the ability to repress TGF-β signaling. Amino-terminal to the Evi-1a, Evi-1c harbors an additional sequence similar to the PR domain, which was originally identified in PRDI-BF1 and RIZ1. In contrast to frequent upregulation of Evi-1a in myeloid malignancies, expression of Evi-1c widely varies depending on cases. Thus, although several lines of evidence suggest that Evi-1a is involved in leukemic transformation of hematopoietic cells, a role for Evi-1c in leukemogenesis has remained elusive. To elucidate the mechanism that underlies leukemogenesis by Evi-1, we investigated the ability of Evi-1 proteins to form oligomeric complexes. We found that Evi-1a forms a homo-oligomer in mammalian cells, whereas Evi-1c exclusively exists as a monomer. Remarkably, Evi-1c has lost the ability to interact with CtBP and failed to efficiently repress TGF-β signaling. We previously reported that AML1-Evi-1, a chimeric transcription factor generated in t(3;21) leukemia, interacts with CtBP as Evi-1a does. In the current study, we also found that AML1-Evi-1 forms a homo-oligomer, suggesting a close relationship between oligomer formation and CtBP binding. Taken together, these data indicate that homo-oligomerization may contribute to the oncogenic potential of the Evi-1 proteins by regulating the interaction with CtBP. These results also identify a novel function of PR domain to dictate oligomerization of transcription factors.

Description: Evi1 (ecotropic viral integration site 1) is essential for proliferation of hematopoietic stem cells and implicated in the development of myeloid disorders. Particularly, high Evi1 expression defines one of the largest clusters in acute myeloid leukemia and is significantly associated with extremely poor prognosis. However, mechanistic basis of Evi1-mediated leukemogenesis has not been fully elucidated. Here, we show that Evi1 directly represses phosphatase and tensin homologue deleted on chromosome 10 (PTEN) transcription in the murine bone marrow, which leads to activation of AKT/mammalian target of rapamycin (mTOR) signaling. In a murine bone marrow transplantation model, Evi1 leukemia showed modestly increased sensitivity to an mTOR inhibitor rapamycin. Furthermore, we found that Evi1 binds to several polycomb group proteins and recruits polycomb repressive complexes for PTEN down-regulation, which shows a novel epigenetic mechanism of AKT/mTOR activation in leukemia. Expression analyses and ChIPassays with human samples indicate that our findings in mice models are recapitulated in human leukemic cells. Dependence of Evi1-expressing leukemic cells on AKT/mTOR signaling provides the first example of targeted therapeutic modalities that suppress the leukemogenic activity of Evi1. The PTEN/AKT/mTOR signaling pathway and the Evi1-polycomb interaction can be promising therapeutic targets for leukemia with activated Evi1.