Inhibitors of RAD51 as potential novel therapies in Acute Myeloid Leukemia Free

RAD51 is a highly conserved gene whose protein product, RAD51, is instrumental in the maintenance of genome integrity in eukaryotes. RAD51 is well known to function in homologous recombination repair ('HRR') in conjunction with the BRCA1 and BRCA2 gene products. It also has other less well-known functions including protection from replication stress ('RS'). RS occurs downstream of incessant oncogene signaling and is therefore typical of cancers in general, and leukemias in particular, but not normal hematopoietic cells. RS is defined by stalled replication forks, which, if unprotected, are susceptible to endonuclease-mediated digestion leading to potentially lethal double strand DNA breaks. RAD51 gathers around stalled forks and protects them by a variety of processes including fork reversal to allow survival and proliferation of cancer cells. RAD51 is, therefore, a potentially important target in leukemias: its inhibition has potential to selectively kill malignant cells.

We have shown that lead compounds of the IBR family of RAD51 inhibitors (IBR2, IBR120 and JKYN-1) have low micromolar single agent activity in a variety of cancer cell lines including representatives of both acute ('AML') and chronic ('CML') myeloid leukemia. Furthermore, these compounds often exhibit strong synergy and at least additivity in combination with both on-label and off-label use of FDA-approved compounds, as well as investigational molecularly-targeted agents, in relevant cell lines; these include FLT3 inhibitors quizartinib, gilteritinib and tuspetinib ((in MV-4-11 and HL60 cell lines) and BCR/ABL inhibitors (imatinib and regorafenib) in the K562 cell line (AACR 2025). Interestingly, strong synergy was demonstrated with JKYN1 and quizartinib, and separately, tuspetinib, in the FLT3 WT line HL60, but only additivity between JKYN-1 and gilteritinib in that cell line; all three of these compounds were extremely potent in the FLT3 mutated MV-4-11 cell line; additivity with JKYN-1 was nonetheless demonstrable, but not synergy. IBR2 exhibited low micromolar single agent activity in the K562 CML line and synergized with both imatinib and (separately) the off-label agent regorafenib. Regorafenib was also strongly synergistic with IBR2 in multiple other solid tumor cell lines, both within and beyond its label in colorectal cancer.

We have confirmed the IBRs inhibit multiple RAD51 functions including hydroxyurea-induced RAD51 focus formation, HRR by DR-GFP assay, and RS protection by RPA focus formation and a DNA fibre assay. They markedly reduce intracellular RAD51 levels. All the IBRs tested to date bind RAD51 strongly by surface plasmon resonance (‘SPR‘), especially JKYN-1, and JKYN-1 is more potent in this and other assays than the standard classical RAD51 inhibitor B02. Our hypothesis is that both the single agent activity and synergy is due primarily to deprotection of RS.

JKYN-1 is a partially optimized (more soluble and more potent) version of IBR120 but is not resistant to degradation by liver microsomes or sufficiently capable of entry into target cells. We have therefore selected 15-20 IBR analogues amongst a pool of ~35,000 created by generative chemistry (InSilico Medicine, Boston) for synthesis and in vitro testing. Anti-leukemic properties of these and our current IBRs will be tested on primary patient derived cells from genetically diverse AML patients. The assay will target stemness properties of AML patients in a high throughput screen designed to provide clinically actionable data for relapsed AML. Treatment dose and combinations will be pre-screened using a highly predictive cell line as a surrogate of human cancer stem cells for subsequently AML patient panels of leukemic stem cells to be examined. These studies will focus on single agent anti-proliferative activity and synergy with a suite of standard and investigational antileukemic drugs including cytosine arabinoside, daunorubicin, quizartinib, gilteritinib, tuspetinib, venetoclax, and azacitadine.