INITIAL EVALUATION OF NOVEL DUAL PIM/PI3K AND TRIPLE PIM/PI3K/MTOR INHIBITORS IN MULTIPLE MYELOMA
Author(s): ,
Mairead Reidy, PhD
Affiliations:
Apoptosis Research Center,National University of Ireland, Galway,Galway,Ireland
,
Marianne vanDijk, PhD
Affiliations:
Apoptosis Research Center,National University of Ireland, Galway,Galway,Ireland
,
Niamh Keane, MB BCh BAO
Affiliations:
Apoptosis Research Center,National University of Ireland, Galway,Galway,Ireland;Haematology,National University of Ireland, Galway,Galway,Ireland
,
Michael O'Neill, PhD
Affiliations:
Inflection Biosciences,Dublin,Ireland
Michael E O'Dwyer MD, FRCPI, FRCPath
Affiliations:
Haematology,National University of Ireland, Galway,Galway,Ireland;Apoptosis Research Center,National University of Ireland, Galway,Galway,Ireland
(Abstract release date: May 21, 2015) EHA Learning Center. Reidy M. Jun 12, 2015; 100316
Disclosure(s): National University of Ireland, Galway
NCBES
Dr. Mairead Reidy
Dr. Mairead Reidy

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Abstract
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Abstract: E1237

Type: Eposter Presentation

Background
Multiple myeloma (MM) is characterised by clonal expansion of malignant plasma cells in the bone marrow (BM). Despite significant advances in treatment it remains incurable. This is largely due to the supportive role the BM environment plays in migration, survival, proliferation and drug resistance.

BM microenvironmental signalling along with other factors such as treatment with proteasome inhibitors (PI) can contribute to activation of the PI3K/AKT survival pathway. Multiple small-molecule inhibitors have been developed to target PI3K/AKT or mTOR kinases, but the efficacy of these drugs is likely to be compromised by the stimulation of compensatory signalling pathways. The redundancy of signalling pathways provides back-up mechanisms allowing escape from targeted inhibition. One such compensatory pathway is that driven by PIM kinases, which produce parallel oncogenic signals to AKT and mTOR and share several downstream molecular targets. 

As with PI3K/AKT, the BM microenvironment plays a major role in PIM activation. PIM1 and particularly PIM2 are known to be highly expressed in MM and play important roles in regulating MYC-driven transcription, apoptosis, cytokine signalling, cell proliferation and protein translation.  Combinations of separate PI3K and PIM inhibitors have shown evidence of synergy in MM cell lines and animal models and a PIM kinase inhibitor has recently shown activity in relapsed/refractory MM.



Aims
We wished to evaluate the activity of a novel family of kinase inhibitors capable of inhibiting not only PIM kinases but also PI3K/AKT (dual inhibitors) and PI3K/AKT/mTOR (triple inhibitors).   

Methods

We evaluated the in-vitro activities of a single pan-PIM (pPIMi), dual PIM/PI3K (IBL-202) and triple PIM/PI3K/mTOR (IBL-301) inhibitor in a number of MM cell lines alongside the pan-PI3K inhibitor GDC-0941 and the pan-PIM inhibitor AZD1208. IBL-202 and IBL-301 are low nanomolar pan-PIM/PI3K and pan-PIM/PI3K/mTOR inhibitors respectively. These dual and triple inhibitors show excellent kinase selectivity profile against a panel of 456 kinases.  Cell viability was assessed using the Cell-Titre Glo assay and apoptosis was determined using Annexin-V/PI staining.  To examine the impact of the microenvironment on the efficacy of these compounds MM cells were co-cultured with HS-5 stromal cells or in a hypoxic glove box (1% O2).



Results
Inhibiting PIM kinases and the PI3K pathway simultaneously using IBL-202 was significantly more potent than a pan-PI3K inhibitor, GDC-0941 alone or pan PIM kinase inhibitors pPIMi or AZD1208. This was observed in all MM cell lines tested and in addition for the compound IBL-301, a triple inhibitor of PIM kinases, PI3K and mTOR. IC50 values were in the 0.5µM and 0.05µM range for IBL-202 and IBL-301, respectively. In comparison pPIMi and GDC-0941 scored an IC50 value between 5 and 10µM while the IC50 for AZD1208 was >20µM (Figure 1).  In addition IBL-202 and IBL-301 caused a reduction in pAkt and known PIM targets, pBad and pS6. Bortezomib caused a marked increase in levels of PIM2 in MM cell lines. A combined treatment of IBL-202 or IBL-301 with bortezomib resulted in a synergistic effect in these cell lines (CI<1).  

In an effort to mimic the tumour microenvironment MM cell lines were co-cultured with the stromal cell line HS-5s. We observed strong induction of PIM2 in MM cells following co-culture.  Co-culture protected MM cell lines against bortezomib-induced cell death, while promoting the apoptotic effect of both IBL-202 and IBL-301 with an increase in Annexin V positive cells from 15% to 40%. This suggests that microenvironmental stimulation could potentially induce synthetic lethality in the presence of these inhibitors. The tumour microenvironment in MM patients is hypoxic compared to healthy controls. Hypoxic conditions led to an increase in levels of PIM1 which is reported to regulate the surface expression of the CXCR4 receptor. The CXCR4 receptor is important for MM cell survival, migration and microenvironment interaction. We have observed a decrease in levels of CXCR4 following treatment with IBL-202 in a dose dependent manner.

The dual and triple inhibitors are optimized with respect to their in vitro ADME properties and have excellent oral bioavailability. In-vivo IBL-301 has been well tolerated, with no signs of toxicity even 20 times above the efficacious dose in a transgenic (KRASV12NSCLC) mouse model.  Testing of IBL-202 in a relevant MM mouse model is planned in the near future.



Summary
IBL-201 and IBL-301 show promising activity in MM cellular models with increased potency compared to inhibitors targeting PIM or PI3K alone and warrant further evaluation in this disease.

Keyword(s): Multiple myeloma, PI3-K/AKT, Pim-1

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