Oral Presentation 32nd Lorne Cancer 2020

Unexpected tumour heterogeneity in CLL relapse on venetoclax revealed by single cell analysis (#2)

Rachel Thijssen 1 2 , Luyi Tian 1 2 , Christoffer Flensburg 1 2 , Charis E Teh 1 2 , Mary Ann Anderson 1 2 3 4 , Piers Blombery 3 4 , Daniel H.D. Gray 1 2 , Ian J. Majewski 1 2 , Matt E. Ritchie 1 2 , Andrew W. Roberts 1 2 3 4 , David C.S. Huang 1 2
  1. The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia
  2. University of Melbourne, Melbourne, VIC, Australia
  3. Royal Melbourne Hospital, Melbourne, VIC, Australia
  4. Peter MacCallum Cancer Centre, Melbourne, VIC, Australia

BCL2 inhibition with venetoclax has proven to be remarkably effective for treating patients with chronic lymphocytic leukaemia (CLL). However, the disease will eventually progress in most patients after years on treatment and further treatment options are limited. Thus, we need to fully define the molecular mechanisms driving venetoclax resistance.

Using paired samples from 20 CLL patients who have progressed while on venetoclax, all the relapse samples were less sensitive to the drug indicating that cell intrinsic changes must drive acquired resistance. One such change is the G101V mutation in BCL2 which blocks venetoclax binding (Blombery et al. Cancer Discov 2019). However, cells bearing this mutation only account for a fraction of the tumor cells at disease progression, strongly implicating other mechanisms that must subvert the action of venetoclax.

We applied the state-of-the art technology - Cellular indexing of transcriptomes and epitopes in single cells (CITE-seq) - which allows us to fully characterize the tumour cells on the basis of proteomics data and gene expression data, from short-read and long-read scRNA-seq. We have identified other drivers of venetoclax resistance: intriguingly, high BCLxL gene expression marked a distinct sub-population of tumor cells from a cluster harboring the BCL2 G101V mutation in one patient. These have unique transcriptional signatures. Using mass spectrometry (Cytometry by Time of Flight; CyTOF), we have now detected high BCLxL in 5 out of 15 patients. In these, there was no evidence for BCLxL amplification or mutation in its 5’ regulatory region previously described in resistant mantle cell lymphoma (Agarwal et al. Nat Med 2019). 1 patient had MCL1 amplification and markedly elevated MCL1 protein expression.

Taken together, our findings reveal that multiple mechanisms must operate to allow acquisition of venetoclax resistance. A detailed understanding of how venetoclax might fail is important to developing strategies to avoid emergence of resistance and to optimally use these powerful anti-cancer agents up front.