Drug tolerant persisters and immunotherapy persister cells exhibit cross-resistance and share common survival mechanisms
Persisters are a rare sub-population of tumor cells that survive anti-cancer therapy and are thought to be a major cause of recurrence. These cells have been identified following both drug- and immune-therapy but are generally considered to be distinct entities. Since both pharmacological agents and immune cells often kill via apoptosis, we tested a hypothesis that both types of cells survive based on reduced mitochondrial apoptotic sensitivity, which in turn would yield a similar and reciprocal multi-agent resistant phenotype. Supporting this hypothesis, we indeed observed that IPCs acquired a reduced sensitivity to multiple drug classes and radiotherapy, suggesting non-immune mechanisms are important in the survival of cancer cells after immunotherapy. Likewise, DTPs developed not only a reduced sensitivity to multiple drug classes and radiotherapy, but also acquired a reduced sensitivity to T cell killing. Both IPCs and DTPs developed a decreased sensitivity to mitochondrial apoptosis. A sub-population of IPCs downregulated  antigen and upregulated PD-L1. Intriguingly, in the IPCs that didn't employ these mechanisms of resistance, a greater decrease in sensitivity to mitochondrial apoptosis was observed, suggesting that the presence or absence of a resistance mechanism can exert selective pressures over the emergence of others. Targeting anti-apoptotic dependencies in persisters increased sensitivity to chemotherapy or CAR T therapy. These results suggest that common biological mechanisms underly survival of persisters, whether derived from immune or drug therapy, and offer an explanation for the acquired cross-resistance to these two types of therapies often observed in the clinic. Overall design: RNA was extracted from parental HeLa cells, selected HeLa immune persister cells (IPC) and 3 different HeLa drug tolerant persister (DTP) cells following respective treatment with IC90 concentrations of 5-FU, Oxaliplatin and Etopiside for 7 days. RNA was extracted using the RNeasy Mini Kit (Qiagen). Prior to RNA sequencing, RNA was treated with DNase using RNase-Free DNase Set (Qiagen). rRNA depletion was performed from 100ng of purified RNA using QIAseq FastSelect rRNA HMR reagents according to manufacturer's protocol. Libraries were prepared using Roche Kapa Biosystems RNA HyperPrep sample preparation reagents on a Beckman Coulter Biomek i7. Finished dsDNA libraries were quantified by Qubit fluorometer and Agilent TapeStation 4200. Uniquely dual indexed libraries were pooled in an equimolar ratio and shallowly sequenced on an Illumina MiSeq to further evaluate library quality and pool balance. The final pool was sequenced with paired-end 150bp reads on an Illumina NovaSeq 6000at the Dana-Farber Cancer Institute Molecular Biology Core Facilities. Sequenced reads were aligned to the UCSC hg38 reference genome assembly and gene counts were quantified using STAR (v2.7.3a).