Anti-CHMP5 single chain variable fragment antibody retrovirus infection induces programmed cell death of AML leukemic cells in vitro

Abstract

Aim: Over-expressed CHMP5 was found to act as oncogene that probably participated in leukemogenesis. In this study, we constructed the CHMP5 single chain variable fragment antibody (CHMP5-scFv) retrovirus and studied the changes of programmed cell death (PCD) of AML leukemic cells after infection by the retrovirus. Methods: The anti-CHMP5 KC14 hybridoma cell line was constructed to generate monoclonal antibody of CHMP5. The protein expression of CHMP5 was studied using immunofluorescence analysis. pMIG-CHMP5 scFv antibody expressible retroviral vector was constructed to prepare CHMP5-scFv retrovirus. AML leukemic U937 cells were infected with the retrovirus, and programmed cell death was studied using confocal microscope, FCM and Western blot. Results: We obtained a monoclonal antibody of CHMP5, and found the expression of CHMP5 was up-regulated in the leukemic cells. After U937 cells were infected with CHMP5-scFv retrovirus, CHMP5 protein was neutralized. Moreover, the infection resulted in a significant increase in apoptosis and necrosis of U937 cells. In U937 cells infected with CHMP5-scFv retrovirus, apoptosis-inducing factor (AIF)-mediated caspase-independent necrotic PCD was activated, but autophagic programmed cell death was not observed. Neither the intrinsic nor extrinsic apoptotic PCD pathway was activated. The granzyme B/perforin-mediated caspase-dependent apoptotic PCD pathway was not activated. Conclusion: CHMP5-scFv retrovirus can neutralize the abnormally high levels of the CHMP5 protein in the cytosol of AML leukemic U937 cells, thereby inducing the programmed cell death of the leukemic cells via AIF-mediated caspase-independent necrosis and apoptosis.

The ESCRT protein CHMP5 promotes T cell leukemia by controlling BRD4-p300-dependent transcription

“…Loss of CHMP5 significantly depleted LICs in vivo suggesting that therapeutic targeting of CHMP5 can achieve more durable T-ALL suppression. Interestingly, high CHMP5 expression has also been reported in hepatocellular carcinoma 81 , and AML 82,83 where its loss correlated with increased apoptosis 84,85 .Whether CHMP5 also regulates AML, in which the BRD4-dependent MYC super-enhancer is also a therapeutic vulnerability 52,86 , and other cancers that display a p300-BRD4 dependency remains to be determined. Therefore, future studies to elucidate CHMP5 function in these malignancies have the potential to significantly advance our understanding of transcriptional addiction mechanisms in cancer cells.…”

“…Loss of CHMP5 significantly depleted LICs in vivo suggesting that therapeutic targeting of CHMP5 can achieve more durable T-ALL suppression. Interestingly, high CHMP5 expression has also been reported in hepatocellular carcinoma 81 , and AML 82,83 where its loss correlated with increased apoptosis 84,85 .…”

A Gene Signature Comprising Seven Pyroptosis-Related Genes Predicts Prognosis in Pediatric Patients with Acute Myeloid Leukemia

“…Previous reports have identified the role of IRF2 in cell proliferation and apoptosis [43, 44], as well as in the development and prognosis of AML in patients [45, 46]. CHMP5, which is a constituent of the ESCRT-III protein complex, along with CHMP3 and CHMP2A, participates in programmed cell death [47]; its levels are correlated with the prognosis of patients with AML [48, 49]. Although there are only a few reports regarding the role of CHMP3 in AML, ESCRT-III-mediated membrane repair is important for pyroptosis and tumor resistance [50].…”

Charged MVB protein 5 is involved in T-cell receptor signaling

“… 8 , 9 , 10 , 11 , 12 CHMP5-knockdown leukemic cells exhibited activation of two programmed cell death pathways: the Granzyme B/Perforin apoptotic pathway and the AIF (apoptosis-inducing factor)-mediated caspase-independent necrosis pathway. 9 Moreover, anti-CHMP5 single chain variable fragment antibody retrovirus infection induces programmed cell death in leukemic cells via AIF-mediated caspase-independent necrosis and apoptosis 13 ; this result suggests that CHMP5 may be involved in cellular apoptotic processes. In addition, CHMP5 is involved in the primary switch that initiates the antiviral mechanism via the regulation of the ISGylation of CHMP2A and CHMP6 and in the availability of the co-activator protein LIP5 to the ESCRT-III-Vps4 complex.…”