We chose to investigate ON 01910.Na because of its reported activity against MCL cell lines through inhibition of the PI3K pathway (23
) and its promising clinical activity in MDS, for which it has now entered a pivotal phase III study (NCT01241500). In initial experiments, we observed rapid induction of apoptosis selectively in CLL cells, while T cells and normal B cells were not or only minimally affected. We were surprised by the strong proapoptotic effect of ON 01910.Na given reports of only minimal apoptosis in response to other clinical grade PI3K inhibitors (11
). Based on its unique chemical structure and mechanism of kinase inhibition (20
), we considered that ON 01910.Na may have additional effects. We chose to investigate the molecular effects and resultant cellular stress response using global gene expression profiling because this approach does not require prior assumptions. The transcriptional profile in ON 01910.Na treated CLL cells was consistent with inhibition of PI3K signaling. In addition we observed upregulation of gene signatures indicating a ROS induced oxidative stress response. We went on to confirm a key role of ROS in ON 01910.Na induced apoptosis in CLL cells: first, we demonstrated a significant accumulation of ROS in response to drug that paralleled onset of apoptosis. Second, the antioxidant NAC effectively antagonized ON 01910.Na mediated apoptosis. Third, we found that ON 01910.Na treatment induced a classic ROS triggered stress response pathway involving activation of pSAP/JNK, nuclear accumulation of c-JUN, and induction of ATF3 and NOXA in all CLL samples tested. It is notable that this stress response differs in some aspects from the NRF2 dominated response we described in MCL cells treated with the proteasome inhibitor bortezomib (33
). NRF2, a key regulator of the protective antioxidant response, is in part regulated through PI3K. The combination of a PI3K inhibitor with a ROS inducing agent has been shown to inhibit the translocation of NRF2 to the nucleus and block upregulation of NRF2 target genes (37
). In keeping with this, ON 1910.Na treated cells mounted no significant NRF2 response (FDR>0.2, data not shown).
Pharmacologic induction of ROS is increasingly recognized as a therapeutic principle to selectively kill transformed cells (39
). Cancer cells are particularly sensitive to disruption of redox homeostasis; a potential vulnerability thought to be due to increased ROS levels originating from oncogene activation (39
). One of the first agents shown to have selective anti-tumor activity through upregulation of ROS is beta-phenylethyl isothiocyanate (PEITC) (41
). This compound was subsequently shown to be effective against fludarabine-resistant CLL cells (42
). Similarly, apoptosis induced in CLL cells by adaphostin, a tyrosine kinase inhibitor of the tyrphostin class (43
), has been shown to depend on the induction of ROS. In vitro, these agents remain active against chemotherapy-resistant CLL cells with dysfunctional TP53; an effect that seems to be primarily due to the induction of NOXA in response to ROS (32
). Despite their encouraging in-vitro activity, these agents have no established clinical benefit in CLL; adaphostin has not entered clinical testing, and a phase I study of PEITC is only about to begin (NCT00968461). In contrast, ON 01910.Na has recently entered a pivotal phase III clinical trials in MDS and is in phase I/II studies for other indications. Interestingly, ON 01910.Na has been reported to be selectively cytotoxic for the myeloid tumor cells without affecting normal hematopoiesis (44
). While induction of oxidative stress has not been described as a therapeutic principle in MDS, ROS plays an important role in the pathogenesis and is increased in MDS cells compared to normal bone marrow cells (reviewed in (45
)). Given our findings, it thus appears plausible that generation of ROS contributes to the selective anti-tumor activity of ON 01910.Na in MDS.
Consistent with a report that ON 01910.Na inhibits PI3K signaling we detected a transcriptional response in drug treated CLL cells indicative of substantial PI3K inhibition. To do this, we selected two representative patients for gene expression analysis and exposed PBMCs to ON 01910.Na in a dose and time dependent fashion. In this way we generated 4 replicates of drug treated cells in each patient that also incorporate dose and time effects. Gene expression in treated cells was compared to untreated cells obtained at the same time points. We then tested whether ON 01910.Na induces the gene expression fingerprint of a PI3K inhibitor using GSEA to estimate the connection of ON 01910.Na induced gene expression changes with previously established signatures of the pan-PI3K inhibitor LY 294002. This approach termed “connectivity mapping” has been developed and used successfully to identify compounds affecting the same cellular pathways (46
). Indeed, the connection between the gene signatures of LY 0294002 and ON 01910.Na was highly significant (FDR 0.01 for downregulated genes; FDR 0.17 for upregulated genes; Supplementary Table S1
, ). Further supporting “on target” effects are the upregulation of genes controlled by FOXO. Transcription factors of the FOXO family are retained in the cytoplasm by PI3K and/or ERK dependent phosphorylation. Upon downregulation of PI3K activity, unphosphorylated FOXO translocates to the nucleus and upregulates numerous genes involved in cell cycle arrest, apoptosis, and stress response (47
). Finally, we also observed downregulation of a subset of genes that we have previously identified as target genes of BCR signaling in CLL cells (7
). Possible reasons why we observed only a partial downregulation of the BCR signature include PI3K independent regulation of some BCR target genes and incomplete inhibition of the pathway by ON 01910.Na, which has been reported to primarily inhibit PI3Kα and PI3Kβ (23
), while BCR signaling activates PI3Kδ (11
). As reported by Prasad and colleagues, we did observe a decrease in pAKT in most samples (), while effects on p-mTOR, pS6K, and p4EBP1 were apparent in some but not other CLL samples tested (data not shown). These differences may be due to the former study testing only two cell lines, or could reflect differences between cell lines and primary samples.
The tumor microenvironment is increasingly recognized as a significant factor contributing to treatment resistance in CLL (8
). Strategies to target cells in the protective environment are therefore a priority. ON 01910.Na combines two complementary activities that could endow it with significant clinical activity. ROS activation can induce apoptosis, particularly in cells with high baseline ROS levels. CLL cells are reported to have high baseline ROS levels (42
) and some of the key signals activated in the secondary lymphoid structures including BCR and CD40 signaling further increase ROS (48
). In addition to increasing oxidative stress through inactivation of NRF2, PI3K inhibition may combine with ROS to target MCL-1, an important survival factor upregulated by stroma contact. MCL-1 protein stability is increased through phosphorylation by AKT (14
) and glutathionylation (42
). Thus, ROS may destabilize MCL-1 through depletion of glutathione while PI3K inhibition blocks the effect of stroma (, and (50
)). While, treatment with a pan-PI3K inhibitor is sufficient to reverse the increase in CLL viability observed in co-culture with stroma cells (50
), or conferred by CD44 activation (17
), it alone does not induce significant apoptosis. In contrast, ON 01910.Na not only blocked the pro-survival effect of HK cells but induced apoptosis in >50% of cells even in the presence of stroma (). ON 01901.Na is also more cytotoxic in vitro than more specific inhibitors such as CAL-101 (11
) or the BTK inhibitor PCI-32765 (10
) that on average reduce CLL cell viability by 10-20% even at concentrations in excess of what is required to inhibit the specific target. These considerations suggest that the combination of a PI3K inhibitor with an agent inducing ROS may confer synergistic anti-tumor effects.
In summary, our studies identify ON 01910.Na as an agent with important preclinical activity in CLL. Specifically, ON 01910.Na is active against CLL cells with loss of TP53 and against cells sheltered by a protective stroma environment. Our results also provide new insights into the mechanism of action of this drug showing that a combination of ROS induction and inhibition of PI3K signaling can effectively combine to induce apoptosis. Insights generated from these studies help identify biomarkers to correlate with clinical activity. Based on these results and work by Prasad and colleagues (23
) we have initiated the first clinical trial with ON 01901.Na in patients with lymphoid malignancies (NCT008615100).