Response to Bruton's tyrosine kinase inhibitors in aggressive lymphomas linked to chronic selective autophagy.

Diffuse large B cell lymphoma (DLBCL) is an aggressive, profoundly heterogeneous cancer, presenting a challenge for precision medicine. Bruton's tyrosine kinase (BTK) inhibitors block B cell receptor (BCR) signaling and are particularly effective in certain molecular subtypes of DLBCL that rely on chronic active BCR signaling to promote oncogenic NF-κB. The MCD genetic subtype, which often acquires mutations in the BCR subunit, CD79B, and in the innate immune adapter, MYD88L265P, typically resists chemotherapy but responds exceptionally to BTK inhibitors. However, the underlying mechanisms of response to BTK inhibitors are poorly understood. Herein, we find a non-canonical form of chronic selective autophagy in MCD DLBCL that targets ubiquitinated MYD88L265P for degradation in a TBK1-dependent manner. MCD tumors acquire genetic and epigenetic alterations that attenuate this autophagic tumor suppressive pathway. In contrast, BTK inhibitors promote autophagic degradation of MYD88L265P, thus explaining their exceptional clinical benefit in MCD DLBCL.

Comparison of TaqMan real-time PCR and p24 Elisa for quantification of in vitro HIV-1 replication.

In this study, TaqMan PCR was used to assess viral replication of HIV-1 infected cells in vitro. This PCR technique was compared with p24 ELISA as a standard method to monitor HIV-1 replication in cell culture. Hut78 T-lymphoblastoid cells were infected with different titres of HIV-1(IIIb) (MOI 0.05-0.0005). The course of HIV-1 replication was monitored by determination of p24 concentrations by ELISA in cell culture supernatants and by quantitation of HIV-1 gag RNA by TaqMan RT-PCR. Additionally, the number of HIV-1 proviral copies was assessed by TaqMan PCR. Monitoring of HIV-1 replication by p24 ELISA and TaqMan RT-PCR revealed comparable kinetics of infection. Both methods provided similar data on the exponential increase and on plateauing of HIV-1 replication. Furthermore, both methods were equally sensitive. However, a 7 log linearity of TaqMan HIV-1 gag PCR was demonstrated without dilution of the specimen, in contrast to p24 ELISA, where because of its narrow range of detectable p24 concentrations, sample dilution was necessary. Although determination of the number of proviral copies by TaqMan PCR does not measure HIV-1 replication, the kinetics of proviral copy number following in vitro inoculation of cells with HIV-1 was nearly the same as the kinetics of HIV-1 RNA copy numbers. In conclusion, TaqMan real-time RT-PCR was demonstrated as a reliable and sensitive tool to quantify and monitor HIV-1 replication in cell culture. It is suggested, therefore, that this technique be an alternative method to monitor HIV-1 replication in vitro.