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Multiple myeloma (MM) is a Ubiquitin Proteasome System (UPS)-dysfunction disease. We previously reported that high PRAME transcript levels associated with unfavorable progression free survival (PFS) in patients with no bortezomib therapy, and bortezomib-containing regimen significantly improved PFS in patients with high PRAME transcript levels, which indicated that PRAME expression was prognostic for MM patients, and was related to proteasome inhibitor treatment. However, molecular mechanisms underlying the above clinical performance remain unclear. In the present study, MM cell models with PRAME knockdown and overexpression were established, and PRAME was identified to play the role of promoting proliferation in MM cells. P-Akt signaling was found to be activated as PRAME overexpressed. As a substrate recognizing subunit (SRS) of the E3 ubiquitin ligase, PRAME targets substrate proteins and mediates their degradation. CTMP and p21 were found to be the novel targets of PRAME in the Cul2-dependent substrate recognition process. PRAME interacted with and mediated ubiquitination and degradation of CTMP and p21, which led to accumulation of p-Akt and CCND3 proteins, and thus promoted cell proliferation and increased bortezomib sensitivity in MM cells.

BackgroundT lymphocytes in tumor microenvironment play a pivotal role in the anti-tumor immunity, and the memory of T cells contributes to the long-term protection against tumor antigens. Compared to solid tumors, studies focusing on the T-cell differentiation in the acute myeloid leukemia (AML) bone marrow (BM) microenvironment remain limited.Patients and methodsFresh BM specimens collected from 103 adult AML patients at diagnosis and 12 healthy donors (HDs) were tested T-cell differentiation subsets by multi-parameter flow cytometry.ResultsCD4 and CD8 T-cell compartments had different constituted profiles of T-cell differentiated subsets, which was similar between AML patients and HDs. Compared to HDs, AML patients as a whole had a significantly higher proportion of CD8 effector T cells (Teff, P = 0.048). Moreover, the T-cell compartment of AML patients with no DNMT3A mutations skewed toward terminal differentiation at the expense of memory T cells (CD4 Teff: P = 0.034; CD8 Teff: P = 0.030; CD8 memory T: P = 0.017), whereas those with mutated DNMT3A had a decrease in CD8 naïve T (Tn) and CD4 effector memory T cells (Tem) as well as an increase in CD4 central memory T cells (Tcm) (P = 0.037, 0.053 and 0.053). Adverse ELN genetic risk correlated with a lower proportion of CD8 Tn. In addition, the low proportions of CD4 Tem and CD8 Tn independently predicted poorer relapse-free survival (RFS, HR [95%CI]: 5.7 (1.4–22.2), P = 0.017 and 4.8 [1.3–17.4], P = 0.013) and event-free survival (EFS, HR [95% CI]: 3.3 (1.1–9.5), P = 0.029; 4.0 (1.4–11.5), P = 0.010), respectively.ConclusionsAML patients had abnormal profiles of BM T-cell differentiation subsets at diagnosis, which was related to DNMT3A mutations. The low proportions of CD4 Tem and CD8 Tn predicted poor outcomes.

Background: Compared to other immune checkpoint molecules, T cell immunoglobulin domain and mucin domain-3 (TIM-3) is highly expressed on natural killer (NK) cells, but its functional role and prognostic significance in acute myeloid leukemia (AML) remains unclear. This study aims to evaluate the role of TIM-3 expression on the cytotoxic and killing capacity of NK cells and its prognostic significance in AML. Methods: AML public single-cell RNA sequencing (scRNAseq) data were used to analyze the correlation of transcript levels between HAVCR2 (encoding TIM-3) and cytotoxic molecules in NK cells. NK cells from the bone marrows of seven newly diagnosed AML patients and five healthy donors (HDs) were stimulated in vitro and cell-killing activity was evaluated. A total of one hundred and five newly diagnosed adult AML patients and seven HDs were tested the expression of TIM-3 and cytotoxic molecules on the bone marrow NK cells by multi-parameter flow cytometry (MFC). Results: Both scRNAseq and MFC analysis demonstrated that TIM-3 expression on NK cells was positively related to the levels of perforin (PFP) and granzyme B (GZMB) (all p < 0.05) in AML. It was PFP and GZMB but not the TIM-3 level that was related to NK-cell-killing activity against K562 cells (p = 0.027, 0.042 and 0.55). A high frequency of TIM-3+ NK cells predicted poorer relapse-free survival (RFS) and event-free survival (EFS) (p = 0.013 and 0.0074), but was not an independent prognostic factor, whereas low GZMB levels in TIM-3+ NK cells independently predicted poorer RFS (p = 0.0032). Conclusions: TIM-3 expression on NK cells is positively related to PFP and GZMB levels but has no relation to cell-killing activity in AML, and low GZMB levels in TIM-3+ NK cells in the diagnostic bone marrows predicts poor outcomes. This study lays a theoretical foundation for the clinical application of immune checkpoint inhibitor treatment.

Background: The effect of the expression of the newly identified immune checkpoint, T cell immunoglobulin and immunoreceptor tyrosine-based inhibition motif domain (TIGIT) on NK cells in core binding factor-acute myeloid leukemia (CBF-AML) remains to be investigated. Methods: Fresh bone marrow samples from a total of 39 newly diagnosed CBF-AML patients and 25 healthy donors (HDs) were collected for testing the phenotype and function state of total NK, CD56bright, and CD56dim NK cell subsets after in vitro stimulation. Results: The frequencies of TIGIT+ cells in total NK, CD56bright, and CD56dim NK cell subsets had no significant difference between patients and HDs. TNF-α and INF-γ levels were uniformly lower in TIGIT+ cells than the corresponding TIGIT− cells in all HDs, whereas those for TIGIT+ to TIGIT− cells in patients were highly heterogenous; TIGIT expression was not related to PFP and GZMB expression in HDs, whereas it was related to higher intracellular PFP and GZMB levels in patients. Patients’ TIGIT+ NK cells displayed lower K562 cell-killing activity than their TIGIT− NK cells. In addition, high frequencies of TIGIT+ cells in total NK and CD56dim NK cells were associated with poor RFS. Conclusions: TIGIT expression affected the diagnostic bone marrow-sited NK cell function and had prognostic significance in CBF-AML patients.