ABSTRACT
Kaposi's sarcoma-associated herpesvirus (KSHV) causes several malignancies in people with HIV including Kaposi's sarcoma and primary effusion lymphoma (PEL). We have previously shown that PEL cell lines require myeloid cell leukemia-1 (MCL1) to inhibit apoptosis. MCL1 is an oncogene that is amplified in cancers and causes resistance to chemotherapy regimens. MCL1 is thus an attractive target for drug development. The emerging clinical relevance and therapeutic potential of MCL1 motivated us to study the roles of this oncogene in PEL in depth. Using a systems biology approach, we uncovered an unexpected genetic interaction between MCL1 and MARCHF5 indicating that they function in the same pathway. MARCHF5 is an E3 ubiquitin ligase most known for regulating mitochondrial homeostasis and antiviral signaling, but not apoptosis. We thus investigated how MCL1 and MARCHF5 cooperate to promote PEL cell survival. CRISPR knockout (KO) of MARCHF5 in PEL cell lines resulted in a significant increase in apoptosis despite the presence of MCL1. The anti-apoptotic function of MARCHF5 was dependent on its E3 ligase and dimerization activities. Loss of MARCHF5 or inhibition of the 26S proteasome furthermore stabilized the MCL1 antagonist NOXA without affecting levels of MCL1. Interestingly, NOXA KO provides a fitness advantage to PEL cells suggesting that NOXA is the pro-apoptotic signal that necessitates the anti-apoptotic activities of MCL1 and MARCHF5. Finally, endogenous reciprocal co-immunoprecipitation experiments show that MARCHF5 and NOXA are found in the same protein complex. Our findings thus provide the mechanistic link that underlies the genetic interaction between MCL1 and MARCHF5. We propose that MARCHF5 induces the degradation of the MCL1 antagonist NOXA thus reinforcing the pro-survival role of MCL1 in these tumor cells. This newly appreciated interaction of the MCL1 and MARCHF5 oncogenes may be useful to improve the design of combination therapies for KSHV malignancies.
ABSTRACT
Kaposi's sarcoma-associated herpesvirus (KSHV) causes several malignancies in people living with HIV, including primary effusion lymphoma (PEL). PEL cell lines exhibit oncogene addictions to both viral and cellular genes. Using CRISPR screens, we previously identified cellular oncogene addictions in PEL cell lines, including MCL1. MCL1 is a member of the BCL2 family, which functions to prevent intrinsic apoptosis and has been implicated in several cancers. Despite the overlapping functions of the BCL2 family members, PEL cells are dependent only on MCL1, suggesting that MCL1 may have nonredundant functions. To investigate why PEL cells exhibit selective addiction to MCL1, we inactivated the intrinsic apoptosis pathway by engineering BAX/BAK1 double knockout cells. In this context, PEL cells become resistant to MCL1 knockdown or MCL1 inactivation by the MCL1 inhibitor S63845, indicating that the main function of MCL1 in PEL cells is to prevent BAX/BAK1-mediated apoptosis. The selective requirement to MCL1 is due to MCL1 being expressed in excess over the BCL2 family. Ectopic expression of several BCL2 family proteins, as well as the KSHV BCL2 homolog, significantly decreased basal caspase 3/7 activity and buffered against staurosporine-induced apoptosis. Finally, overexpressed BCL2 family members can functionally substitute for MCL1, when it is inhibited by S63845. Together, our data indicate that the expression levels of the BCL2 family likely explain why PEL tumor cells are highly addicted to MCL1. Importantly, our results suggest that caution should be taken when considering MCL1 inhibitors as a monotherapy regimen for PEL because resistance can develop easily. IMPORTANCE Primary effusion lymphoma (PEL) is caused by Kaposi's sarcoma-associated herpesvirus. We showed previously that PEL cell lines require the antiapoptotic protein MCL1 for survival but not the other BCL2 family proteins. This selective dependence on MCL1 is unexpected as the BCL2 family functions similarly in preventing intrinsic apoptosis. Recently, new roles for MCL1 not shared with the BCL2 family have emerged. Here, we show that noncanonical functions of MCL1 are unlikely essential. Instead, MCL1 functions mainly to prevent apoptosis. The specific requirement to MCL1 is due to MCL1 being expressed in excess over the BCL2 family. Consistent with this model, shifting these expression ratios changes the requirement away from MCL1 and toward the dominant BCL2 family gene. Together, our results indicate that although MCL1 is an attractive chemotherapeutic target to treat PEL, careful consideration must be taken, as resistance to MCL1-specific inhibitors easily develops through BCL2 family overexpression.