PROTEASOME INHIBITOR CARFILZOMIB HAS SYNTHETIC LETHALITY WITH RUXOLITINIB IN MYELOPROLIFERATIVE NEOPLASMS
Author(s): ,
Simone Claudiani
Affiliations:
Department of Haematology, Hammersmith Hospital,Imperial College Healthcare NHS Trust,London,United Kingdom;Centre for Haematology,Imperial College London,London,United Kingdom
,
Clint C Mason
Affiliations:
Division of Hematology and Hematologic Malignancies,University of Utah,Salt Lake City,United States
,
Carme Ripoll Fiol
Affiliations:
Centre for Haematology,Imperial College London,London,United Kingdom
,
Katya Mokretar
Affiliations:
Department of Haematology, Hammersmith Hospital,Imperial College Healthcare NHS Trust,London,United Kingdom
,
Dragana Milojkovic
Affiliations:
Department of Haematology, Hammersmith Hospital,Imperial College Healthcare NHS Trust,London,United Kingdom
,
Michael Deininger
Affiliations:
Division of Hematology and Hematologic Malignancies,University of Utah,Salt Lake City,United States
,
Jane Apperley
Affiliations:
Department of Haematology, Hammersmith Hospital,Imperial College Healthcare NHS Trust,London,United Kingdom
Jamshid Khorashad
Affiliations:
Centre for Haematology,Imperial College London,London,United Kingdom
EHA Library. Claudiani S. Jun 15, 2019; 267059; PS1442
Simone Claudiani
Simone Claudiani
Contributions
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Abstract

Abstract: PS1442

Type: Poster Presentation

Presentation during EHA24: On Saturday, June 15, 2019 from 17:30 - 19:00

Location: Poster area

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.

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.

Session topic: 15. Myeloproliferative neoplasms - Biology & Translational Research

Keyword(s): Myeloproliferative disorder, Proteasome inhibitor, Ruxolitinib

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