PS1442 PROTEASOME INHIBITOR CARFILZOMIB HAS SYNTHETIC LETHALITY WITH RUXOLITINIB IN MYELOPROLIFERATIVE NEOPLASMS
Authors: Claudiani S. , Mason C.C. , Fiol C. Ripoll , Mokretar K. , Milojkovic D. , Deininger M. , Apperley J. , Khorashad J. 
Published: June 2019

Background:

Ruxolitinib, like other clinically tested JAK2 inhibitors, is effective in reducing spleen size and symptom burden in patients with myelofibrosis (MF), but in most patients does not significantly reduce the mutant clone or bone marrow fibrosis. Additionally most patients develop resistance after an initial response or require dose reductions due to toxicity. Lastly, given compelling evidence that the microenvironment plays a crucial role in the development of myeloproliferative neoplasms (MPN), targeting the MPN cell clone in isolation may have limited efficacy.

Aims:

Therefore, we aimed to identify the signalling pathways promoting the survival of MPN cells in the presence of Ruxolitinib and the microenvironment.

Methods:

We applied a pooled shRNA library screen of ∼5000 genes (Cellecta, Inc.) to HEL cells, a JAK2 V617F‐mutated MPN cell line model, and we used conditioned medium (CM) from human‐derived stromal cells (HS‐5) to recapitulate in vitro the microenvironment component. Two culture conditions were tested, each run in duplicate: CM + ruxolitinib and RPMI + ruxolitinib. We also performed shRNA library screen for CD34+ cells derived from peripheral blood from a patient with primary MF treated with JAK2‐inhibitor. Primary cells were cultured in CM and the identified candidate shRNAs were compared with those resulting from the cell line screen. Chemical validation of the top‐depleted shRNAs was carried out in vitro in both the HEL cell line and primary CD34+ cells from MF patients using proliferation (MTS) and Trypan‐blue viability assays following culture with the inhibitors.

Results:

shRNAs were considered significant for the cell survival if their depletion was ≥ 2‐fold compared to baseline sample and was observed in at least 2 shRNAs targeting the same gene.

Our screening of the ruxolitinib‐treated HEL cells cultured in CM identified 13 candidate genes with high fold depletion only in presence of the microenvironment, which will be part of future analysis. We decided in first instance to focus our attention on those genes whose depletion was maintained in the presence of the microenvironment. Among these, nucleocytoplasmic transport (confirming the recently published evidence: D Yan et al., 2018) and ubiquitin‐proteasome pathway genes were the most depleted in both settings (with and without CM). Interestingly, some of the proteasome machinery genes were highly depleted also in the primary cell shRNA screen.

The in vitro proliferation and viability assays showed that proteasomal inhibition reduces the viability of both HEL cells and MF CD34+ cells and that the combination of carfilzomib with ruxolitinib is additive or synergistic.

Summary/Conclusion:

The proteasome is crucial to cell response to oxidative stress, which may be induced by JAK/STAT pathway. The latter represents the main pathogenic driver of MPNs. Furthermore, proteasome inhibitors are known to negatively affect the NF‐Kb pathway, which has been shown to be hyperactive in MPN, especially in MF. In view of the above evidence, it is possible to speculate that proteasome inhibition may have a significant role in the lethality of MPN cells, although potential alternative mechanisms responsible for the efficacy of carfilzomib cannot be ruled out.

Although our findings still require a mechanistic explanation, they show for the first time that the combination of JAK2 and proteasome inhibitors increases the lethality of MPN cells and deserves further investigation as a potential therapeutic strategy for MPN patients.