High-Throughput Drug Sensitivity and Resistance Testing (DSRT) Platform Reveals Novel Candidate Drugs For Advanced Phase BCR-ABL1-Positive Leukemia
Authors: Pietarinen Paavo , Majumder Muntasir Mamun , Malani Disha , Murumägi Astrid , Pemovska Tea , Koskenvesa Perttu , Kallioniemi Olli , Wennerberg Krister , Heckman Caroline A. , Mustjoki Satu , Porkka Kimmo 
Published: October 2019

Abstract

Background
Advanced BCR-ABL1-positive leukemias (chronic myeloid leukemia in blast crisis and Ph+ALL) remain a therapy challenge despite advances in tyrosine kinase inhibitor (TKI) therapy. Emergence of primary and secondary resistance due to gatekeeper and compound mutations within the BCR-ABL1 kinase domain is common even with the novel 2nd and 3rd generation TKIs (dasatinib, nilotinib, ponatinib). We set out to identify novel candidate drugs for advanced BCR-ABL1-positive leukemias by using an unbiased high-throughput drug testing platform and utilizing both primary patient cells and cell lines.

Methods
As a study material we used 3 CML cell lines representing different types of CML blast phases. In addition to commonly used K562 cells, EM-2 and MOLM-1 cell lines were tested. AML cell lines (AML-193, AP-1060, HL60ATCC, HL60TB, Kasumi-1, KG-1, ME-1, MOLM-13, MONO-MAC-6, MUTZ-2, MV4-11, NOMO-1, SH-2, SHI-1, SIG-M5, SKM-1, THP-1) were used as cell line controls.
To verify the results obtained from cell lines, primary bone marrow (BM) cells were derived from 2 TKI-resistant CML BC patients. Patient 1 had developed resistance to imatinib and dasatinib due to a T315I mutation, whereas patient 2 was resistant to nilotinib, dasatinib and ponatinib due to a V299L and a compound mutation. BM cells from 4 healthy individuals were used as controls.
The functional profiling of drug responses was performed with a high-throughput drug sensitivity and resistance testing (DSRT) platform comprising of 306 anti-cancer agents (FDA/EMA approved, investigational and experimental compounds). Cells were dispensed to pre-drugged 386-well plates of 5 different concentrations and incubated in a humidified incubator with 5% CO2 at 37 °C for 72 hours. Cell viability was measured by using a luminescent cell viability assay (CellTiter-Glo). From plate reads a Drug Sensitivity Score (DSS) was calculated for each drug as a measure of cytotoxicity.
In addition to DSRT, Human Phospho-Kinase Array Kit (R&D systems) was used to analyze the phosphokinase profile in patient samples.

Results
Based on initial comparisons between CML and AML cells lines, nonspecific cytotoxic drugs, which showed high activity in all cell lines, were omitted from further analysis. The DSS scores from different CML cells lines correlated relatively closely (EM-2 vs. K-562, r=0.89; EM-2 vs. MOLM-1, r=0.82; K-562 vs. MOLM-1, r=0.78; p<0.0001 for all correlations).
We next ranked the DSRT data according to the DSS values with most sensitive drugs showing the highest DSS scores. The primary cells from CML BC were further normalized against the median values from healthy controls, resulting in leukemia-specific sensitivity scores (sDSS). Ranked results from the DSRT analysis are shown in the Table.
As expected, the cell lines were sensitive to TKIs, with the exception of the MOLM-1, which showed only modest sensitivity. The clinically TKI-resistant patient samples were also TKI-resistant ex vivo, further validating the DSRT assay data.
Drugs which showed efficacy in both the cell lines and the TKI-resistant patients included HSP90 inhibitors (NVP-AUY922, BIIB021), a NAMPT inhibitor daporinad and the protein translation inhibitor omacetaxine (homoharringtonine). Phosphokinase antibody array results from the patient samples showed increased expression of the HSP27 protein as a putative biomarker for HSP90 inhibitor response.

Conclusions
DSRT is a powerful assay for identifying novel candidate molecules for refractory BCR-ABL1-positive leukemias. Our results indicate that HSP90 and NAMPT inhibitors in particular warrant further clinical evaluation both by analyzing a larger set of primary patient samples and by performing proof-of-concept clinical studies. The results also pave way for designing rational combination therapy strategies.

Disclosures:
Mustjoki: Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau. Porkka:BMS: Consultancy, Research Funding, Speakers Bureau; Novartis: Consultancy, Research Funding, Speakers Bureau.