Synthetic Lethal RNAi Screen of the Human Kinome with Cytarabine in Myeloid Leukemias.
Authors: Tibes Raoul , Bogenberger James , Hagelstrom Tanner , Buechel Megan E. , Bhagavatula Keertivibha , Choudhary Ashish , Azrosa David 
Published: October 2019

Abstract
Abstract 590

Background:
To improve response and increase the number of patients responding to Ara-C, new genes/mechanisms/pathways need to be discovered that underlie and determine the sensitivity to Ara-C. We hypothesized that applying synthetic lethal RNAi screens of >570 kinases of the human kinome +/- Ara-C would be a fast and unbiased way to identify kinases that modulate sensitivity to Ara-C in myeloid cells.

Methods:
First, we developed a High-Throughput RNAi (HT-RNAi) platform for transient siRNA gene silencing in myeloid cells. Using lipid-based reverse transfection, TF1 and THP-1 myeloid cells were incubated with 2 different siRNA sequences per gene targeting 572 kinases (Tibes, EHA 2009). At 48 hrs, Ara-C at an IC30 dose was added and proliferation/viability was measured using a luminescence-based assay 48 hours after Ara-C treatment. Additionally, to assess high- vs. low dose Ara-C, a screen at an IC70 dose of Ara-C was performed in TF1 cells. Screens were performed in duplicate. The log2 ratio of each individual treatment of si+Ara-C over si alone was calculated [siRNA+drug : siRNA]. Hits were defined as the log2 ratio of individual siRNA >2 standard deviation (STD) below the median of all siRNA treatments (log2 ratios) on an individual plate or for the entire screen, respectively.

Results:
Transfection efficacy, determined by a universal lethal siRNA vs. non-silencing and buffer control, was between 75-80% for TF1 and >85% for THP-l. Correlation was high, i.e. for THP-1 (same Ara-C dose) with R2=0.67 for duplicate screens. Data was first analyzed to identify reproducible hits across independent duplicate RNAi screens for each cell line. Hits were defined of at least one same sequence siRNA reducing viability in combination with Ara-C in both (duplicate) screens to greater than 2 STD compared to siRNA or Ara-C alone (see also Methods). For TF1, 9 genes (1.6%) met these criteria. The top hit in TF1 was ATR for which all 4/4 siRNA sensitized to Ara-C; 4 genes each were positive for 3/4 and 2/4 siRNA. For THP-1, 8 genes were hits (7 genes with 2/4 siRNA and 1 gene with 3/4 siRNA) representing 1.4% of the entire screen. Strong hits in TF1 included ABL-1 and CHEK1 and for THP-1, ABL-2 and CHEK1. Apart from the top hit CHEK1, only one other gene reduced proliferation/viability in combination with Ara-C across both cell lines and thus, may be a novel universal sensitizer to Ara-C. This gene is centrally involved in regulating trafficking of membrane and receptor kinases of the MAPK family/pathways.
Comparing high- vs. low-dose Ara-C, 4 genes exhibited sensitization to Ara-C at low concentrations only, and 10 genes at high Ara-C concentrations (each 2/2 si). Genes sensitizing at low Ara-C are involved in cell-cycle (3 of 4 genes); at high Ara-C, KEGG pathway analysis showed enrichment for purine, pyrimidine, IGF and MAPK pathways.
In secondary functional validation screens using 4 siRNA sequences, CHEK1 has validated, whereas cell-cycle regulatory genes (i.e. AURKB) have not to date. In preliminary experiments, treatment of myeloid cells with CHEK1 inhibitors sensitized to Ara-C. A list of high-priority candidate targets is assembled and targets are being validated.

Conclusions:
Herein, we present the first large-scale kinase RNAi synthetic lethal screen in myeloid cells in combination with Ara-C. Only few kinases, 1-2% of the kinome, were identified whose silencing sensitizes to Ara-C. Two genes were potential universal sensitizers in AML: CHEK1, for which inhibitors are in early clinical development including in AML. We are currently conducting clinical trials with a novel, specific well tolerated CHEK inhibitor plus chemotherapy in solid tumors. Supported by our data we are encouraged to translating this concept into design of combination trials of CHEK1 inhibitor with Ara-C in AML. A potential novel sensitizer gene to Ara-C was identified that regulates kinase membrane trafficking and can activate the MAPK pathway. Validation of this gene is currently ongoing. The initial finding that distinct genes may modulate response at different Ara-C doses is of importance and could have clinical implications in the future.
Functional genomics provide a fast and attractive approach to identify molecular targets and vulnerabilities in AML. RNAi synthetic lethal screens in leukemias hold promise for design of rational combination therapies that can be rapidly translated into the clinic.

Disclosures:
Tibes: Arizona Biomedical Research Commission: Peer reviewed grant funding for the presented project.