Yoichi Imai

The introduction of immunotherapies has led to remarkable progress in the treatment of hematological malignancies, including B-cell malignancies such as B-cell lymphoma and multiple myeloma (MM). Although conventional therapeutic antibodies are effective as immunotherapy for newly diagnosed and relapsed/refractory B-cell lymphoma and MM, some cases are resistant. Chimeric antigen receptor (CAR) T-cell therapies targeting B-cell lymphoma and MM have progressed through several generations, and have improved treatment strategies for relapsed/refractory disease. In addition to conventional therapeutic antibodies, bispecific antibodies targeting both tumor cells and T cells have been developed for MM. Both CAR T-cell therapies and bispecific antibodies are effective for heavily treated patients with relapsed/refractory disease. However, most patients treated with these therapies relapse, and serious adverse events like cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) are problematic. This Progress in Hematology, “Novel treatment strategies for hematological malignancies in the immunotherapy era,” focuses on such limitations and the future outlook for CAR T-cell therapies and bispecific antibodies for B-cell malignancies. The role of NK cells in anti-tumor immunity for AML and various therapeutic strategies for NK-cell therapy in AML is also discussed.

Acute myeloid leukemia (AML) is a hematologic malignancy that frequently relapses, even if remission can be achieved with intensive chemotherapy. One known relapse mechanism is the escape of leukemic cells from immune surveillance. Currently, there is no effective immunotherapy for AML because of the lack of specific antigens. Here, we aimed to elucidate the association between CD155 and CD112 in AML cell lines and primary AML samples and determine the therapeutic response. Briefly, we generated NK-92 cell lines (NK-92) with modified DNAX-associated molecule 1 (DNAM-1) and T-cell immunoglobulin and ITIM domain (TIGIT), which are receptors of CD155 and CD112, respectively. Analysis of 200 cases of AML indicated that the survival of patients with high expression of CD112 was shorter than that of patients with low expression. NK-92 DNAM-1 exhibited enhanced cytotoxic activity against AML cell lines and primary cells derived from patients with AML. DNAM-1 induction in NK-92 cells enhanced the expression of cytotoxicity-related genes, thus overcoming the inhibitory activity of TIGIT. Between CD155 and CD112, CD112 is an especially important target for natural killer (NK)-cell therapy of AML. Using a xenograft model, we confirmed the enhanced antitumor effect of NK-92 DNAM-1 compared with that of NK-92 alone. We also discovered that CD112 (Nectin-2), an immune checkpoint molecule belonging to the Nectin/Nectin-like family, functions as a novel target of immunotherapy. In conclusion, modification of the DNAM-1/CD112 axis in NK cells may be an effective novel immunotherapy for AML. Furthermore, our findings suggest that the levels of expression of these molecules are potential prognostic markers in AML.

Positron emission tomography (PET) is an imaging modality used for the noninvasive assessment of tumor staging and response to therapy. PET with ¹⁸F labeled fluorodeoxyglucose (¹⁸F-FDG PET) is widely used to assess the active and inactive lesions in patients with multiple myeloma (MM). Despite the availability of ¹⁸F-FDG PET for the management of MM, PET imaging is less sensitive than next-generation flow cytometry and sequencing. Therefore, the novel PET radiotracers ⁶⁴Cu-LLP2A, ⁶⁸Ga-pentixafor, and ⁸⁹Zr-daratumumab have been developed to target the cell surface antigens of MM cells. Furthermore, recent studies attempted to visualize the tumor-infiltrating lymphocytes using PET imaging in patients with cancer to investigate their prognostic effect; however, these studies have not yet been performed in MM patients. This review summarizes the recent studies on PET with ¹⁸F-FDG and novel radiotracers for the detection of MM and the resulting preclinical research using MM mouse models and clinical studies. Novel PET technologies may be useful for developing therapeutic strategies for MM in the future.

Masamitsu Mikami, Tatsuya Masuda, Takuya Kanatani, Mina Noura, Katsutsugu Umeda, Hidefumi Hiramatsu, Hirohito Kubota, Tomoo Daifu, Atsushi Iwai, Etsuko Yamamoto Hattori, Kana Furuichi, Saho Takasaki, Sunao Tanaka, Yasuzumi Matsui, Hidemasa Matsuo, Masahiro Hirata, Tatsuki R. Kataoka, Tatsutoshi Nakahata, Yasumichi Kuwahara, Tomoko Iehara, Hajime Hosoi, Yoichi Imai, Junko Takita, Hiroshi Sugiyama, Souichi Adachi, and Yasuhiko Kamikubo. Mol. Cells 2022;45:886-95. https://doi.org/10.14348/molcells.2022.2031

Acute myeloid leukemia (AML) relapse is considered to be related to escape from antitumor immunity. Changes in the expression of immune checkpoints, including B7 homolog (H)1 and B7‑H2, have been reported to contribute to AML progression. Binding of T cell immunoglobulin and immunoreceptor tyrosine‑based inhibitory motif domain (TIGIT) among other immune checkpoints on natural killer (NK) and T cells to CD155/CD112 in tumors is supposed to be inhibitory; however, the mechanism by which changes in CD155 and CD112 expression affect tumor immunity remains unclear. When the increased expression of CD155 and CD112 activates Raf‑MEK‑ERK pathway and Raf‑MEK‑ERK pathway is one of the targets of FMS‑like tyrosine kinase 3 (FLT3) inhibition. The present study investigated the alterations in CD155 and CD112 expression under FLT3 inhibition (quizartinib and gilteritinib) and studied its effect on NK and T cell cytotoxicity. CD155 and CD112 expression was analyzed using flow cytometry and reverse transcription‑quantitative PCR in AML cell lines with or without FLT3 mutation using FLT3 inhibitors. CD155 and CD112 expression was specifically downregulated by FLT3 inhibition in FLT3‑mutated cell lines. Direct cytotoxicity and antibody‑dependent cellular cytotoxicity against these cells by NK cells were enhanced. However, the cytotoxicity of γδ T cells with low TIGIT expression compared with NK cells was not enhanced in direct cytotoxicity assay using luciferase luminescence. The analysis of clinical trials from The Cancer Genome Atlas (TCGA) revealed that high CD155 and CD112 expression is associated with poor overall survival. The enhanced cytotoxicity of NK cells against CD155‑ and CD112‑downregulated cells following FLT3 inhibition indicated CD155 and CD112 as possible targets of immunotherapy for AML using FLT3 inhibitors.

The emergence of tyrosine kinase inhibitors as part of a front‐line treatment has greatly improved the clinical outcome of the patients with Ph⁺ acute lymphoblastic leukemia (ALL). However, a portion of them still become refractory to the therapy mainly through acquiring mutations in the BCR‐ABL1 gene, necessitating a novel strategy to treat tyrosine kinase inhibitor (TKI)‐resistant Ph⁺ ALL cases. In this report, we show evidence that RUNX1 transcription factor stringently controls the expression of BCR‐ABL1, which can strategically be targeted by our novel RUNX inhibitor, Chb‐M'. Through a series of in vitro experiments, we identified that RUNX1 binds to the promoter of BCR and directly transactivates BCR‐ABL1 expression in Ph⁺ ALL cell lines. These cells showed significantly reduced expression of BCR‐ABL1 with suppressed proliferation upon RUNX1 knockdown. Moreover, treatment with Chb‐M' consistently downregulated the expression of BCR‐ABL1 in these cells and this drug was highly effective even in an imatinib‐resistant Ph⁺ ALL cell line. In good agreement with these findings, forced expression of BCR‐ABL1 in these cells conferred relative resistance to Chb‐M'. In addition, in vivo experiments with the Ph⁺ ALL patient‐derived xenograft cells showed similar results. In summary, targeting RUNX1 therapeutically in Ph⁺ ALL cells may lead to overcoming TKI resistance through the transcriptional regulation of BCR‐ABL1. Chb‐M' could be a novel drug for patients with TKI‐resistant refractory Ph⁺ ALL.

Background Multiple myeloma (MM) is an incurable hematological malignancy. Despite the introduction of several novel drugs, most patients relapse. Biomarkers to identify the early signs of relapse will make it possible to adjust the therapeutic strategy before the disease worsens. Although understanding genetic changes is important for the treatment of MM, currently known biomarkers of relapse, including serum free-light chains and monoclonal paraproteins, are not associated with genetic changes. Methods We therefore performed a multicenter study to examine the usefulness of circulating cell-free DNA (cfDNA) present in the peripheral blood (PB) plasma of patients as a biomarker for MM relapse. Results We identified several driver mutations by combined analysis of next-generation sequencing and existing databases of candidate oncogenes. Furthermore, relapse was detected more sensitively by monitoring the circulating cfDNA with these driver mutations than by conventional serum free-light chain examination. Conclusion These results suggest the potential utility of cfDNA in the PB plasma of patients as a relevant early biomarker for MM relapse.

Background: Multiple myeloma (MM) is an incurable disease. The acquisition of resistance to drugs, including immunomodulatory drugs (IMiDs), has a negative effect on its prognosis. Cereblon (CRBN) is a key mediator of the bioactivities of IMiDs such as lenalidomide. Moreover, genetic alteration of CRBN is frequently detected in IMiD-resistant patients and is considered to contribute to IMiD resistance. Thus, overcoming resistance to drugs, including IMiDs, is expected to improve clinical outcomes. Here, we examined potential mechanisms of a histone deacetylase (HDAC) inhibitor and Akt inhibitor in relapsed/refractory MM patients. Methods: We established lenalidomide-resistant cells by knocking down CRBN with RNAi-mediated downregulation or knocking out CRBN using CRISPR-Cas9 in MM cells. Additionally, we derived multi-drug (bortezomib, doxorubicin, or dexamethasone)-resistant cell lines and primary cells from relapsed/refractory MM patients. The effects of HDAC and Akt inhibitors on these drug-resistant MM cells were then observed with a particular focus on whether HDAC inhibitors enhance immunotherapy efficacy. We also investigated the effect of lenalidomide on CRBN-deficient cells. Results: The HDAC inhibitor suppressed the growth of drug-resistant MM cell lines and enhanced the antibody-dependent cellular cytotoxicity (ADCC) of therapeutic antibodies by upregulating natural killer group 2D (NKG2D) ligands in MM cells. CRBN-deficient cells showed lenalidomide-induced upregulation of phosphorylated glycogen synthase kinase-3 (p-GSK-3) and c-Myc phosphorylation. Moreover, HDAC and Akt inhibitors downregulated c-Myc by blocking GSK-3 phosphorylation. HDAC and Akt inhibitors also exhibited synergistic cytotoxic and c-Myc-suppressive effects. The dual HDAC and PI3K inhibitor, CUDC-907, exhibited cytotoxic and immunotherapy-enhancing effects in MM cells, including multi-drug-resistant lines and primary cells from lenalidomide-resistant patients. Conclusions: The combination of an HDAC and an Akt inhibitor represents a promising approach for the treatment of relapsed/refractory MM.

Background: Multiple myeloma (MM) patients may undergo relapse and experience resistance to existing therapies. Cereblon (CRBN) is key mediator of the bioactivities of immunomodulatory drugs (IMiDs), including lenalidomide. Moreover, genetic alteration of CRBN is frequently detected in IMiD-resistant patients and considered to contribute to IMiD resistance. Thus, overcoming resistance to drugs, including IMiDs, is expected to improve clinical outcomes. Here, we examined potential mechanisms of a histone deacetylase (HDAC) inhibitor and Akt inhibitor in treatment of relapsed/refractory MM patients.Methods: We established lenalidomide-resistant cells by knocking down or knocking out CRBN in MM cells. Additionally, we derived multi-drug (bortezomib, doxorubicin, or dexamethasone)-resistant cell lines and primary cells from relapsed/refractory MM patients. The effects of HDAC and Akt inhibitors on these drug-resistant MM cells were then observed with a particular focus placed on whether HDAC inhibitors enhance immunotherapy efficacy. We also investigated the effect of lenalidomide on CRBN-deficient cells.Results: HDAC inhibitor suppressed the growth of drug-resistant MM cell lines, and enhanced antibody-dependent cellular cytotoxicity (ADCC) of therapeutic antibodies by upregulating natural killer group 2D (NKG2D) ligands in MM cells. CRBN-deficient cells showed lenalidomide-induced upregulation of glycogen synthase kinase-3 (p-GSK-3) and c-Myc phosphorylation. Meanwhile, HDAC and Akt inhibitors downregulated c-Myc by blocking GSK-3 phosphorylation. HDAC and Akt inhibitors also exhibited synergistic cytotoxic and c-Myc–suppressive effects. Moreover, the dual HDAC and PI3K inhibitor, CUDC-907, exhibited cytotoxic and immunotherapy-enhancing effects for MM cells, including for multi-drug-resistant lines and primary cells including lenalidomide-resistant patients.Conclusions: Combined HDAC and Akt inhibition represents a promising approach for the treatment of relapsed/refractory MM.