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BET and FLT3 inhibition for FLT3-ITD AML
day of dosing and tumor measurement), PLX51107 treat- ment groups showed 57%, 81%, and 97% tumor growth inhibition (%TGI) at 10, 20, and 40 mg/kg doses, respec- tively. These results indicate that PLX51107 has single agent activity against AML in vivo. No adverse effects were observed at doses of 10 and 20 mg/kg. The high dose (40 mg/kg) was also tolerated but exhibited some toxicity (e.g., body weight loss in some animals) which recovered over time. The toxicity is likely due to the high exposure of PLX51107 at this dose (AUC0-24 = 211,000 ng x h/mL). Based on in-life tolerability and body weight changes, the maximum dose without body weight loss in this study was 20 mg/kg with corresponding AUC0-24 of 78,800 ng x h/mL. In the same model, we tested whether the PLX51107 treatment had any effect of MYC gene expres- sion. Mice were given a single dose of 20 mg/kg PLX51107 and MYC RNA was measured in the tumor tissues after 2, 4 and 8 h (Figure 1B). Relative to pre-dose MYC expres- sion, we observed a 90% decrease in MYC gene expres- sion between 2 and 4 h post dose that only partially rebounded at 8 h post dose.
We next evaluated efficacy of the quizartinib-PLX51107 combination in the MV4-11 xenograft model using two different dosing regimens (PLX51107 at 20 mg/kg was used in both cases). The first experiment examined the effect of PLX51107 in combination with continuous (25 days), low-dose (1 mg/kg) quizartinib (Figure 1C). d0 is first day of dosing. On d5, PLX51107 alone almost com- pletely suppressed tumor growth (TGI=98%) while quizartinib monotherapy and quizartinib-PLX51107 com- bination caused tumor regression (22% and 55%, respec- tively). Tumors in mice in the single agent quizartinib group progressed on d9 whereas mice in the combination group had maintained tumor regression until d26. On d14, 5 out of 7 animals in the quizartinib-PLX51107 group had no measurable tumors (100% decrease) (Figure 1D).
The third MV4-11 xenograft study examined the effect of PLX51107 on the duration of response after short-term (5 days) co-administration with high-dose (5 mg/kg) quizartinib (Figure 1E and F). By d4, PLX51107 alone delayed tumor growth (%TGI=68%) while quizartinib monotherapy and quizartinib-PLX51107 combination caused significant tumor regression (61% and 62%, respectively). The benefit of adding PLX51107 became evident when the durations of response were compared. Mice in the 5 mg/kg quizartinib group progressed on d26 (22 days after the last quizartinib dose) whereas mice in the quizartinib-PLX51107 group maintained tumor regres- sion until d39. Two out of eight animals in the combina- tion group achieved complete remission (CR) by d4 and six achieved CR by d18, while no animal in the single agent groups experienced a CR. Furthermore, tumors in mice that relapsed after short-term, single-agent quizar- tinib treatment decreased in size when challenged with quizartinib + PLX51107 on d49.
Taken together, these studies showed that, in a mouse xenograft model, PLX51107 enhanced the response of AML cells to continuous, low-dose quizartinib treatment and significantly improved the duration of response to short-term, high-dose quizartinib treatment without atten- dant toxicity. In addition, PLX51107 showed activity against AML not controlled by prior quizartinib treatment.
The efficacy observed in the xenograft studies reflects primarily the synergy between two cytotoxic agents act- ing at different levels of oncogenic signaling (i.e., proximal
and downstream) in malignant cells not associated with BM stroma. In the clinical setting, BET inhibition could reduce the level of survival factors such as MYC in blasts on stromal cells, thereby undermining the resistance to the effects of FLT3 inhibition conferred by the microenviron- ment. Before we could test this hypothesis using co-cul- ture of primary blast samples with mesenchymal stromal cells, it was necessary to determine the concentration of PLX51107 in culture medium that would give rise to a pharmacodynamic effect comparable to that observed in humans at clinically achievable drug exposure. To that end, we developed a surrogate assay modeled on the one we had previously used to estimate FLT3 inhibition in vivo.22 A phase I dose escalation study of PLX51107 in patients with solid tumors and hematologic malignancy (clinicaltrials.gov identifier: NCT02683395) is underway, and the results of this trial will be published separately. Plasma samples collected from patients on this study were incubated with OCI-AML3 cells (NPM1-mutated, FLT3- wild-type). The degree and duration of MYC inhibition were quantified by immunoblotting and densitometry. We refer to this value as the plasma inhibitory activity for MYC (or MYC PIA). The OCI-AML3 cell line was selected because MYC expression is suppressed by PLX51107 (Figure 2A and B) but not by quizartinib (Figure 2C). Such specificity makes it possible to use the developed assay to measure the BET-mediated (FLT3-independent) effect in a quizartinib-PLX51107 combination trial.
Six dose levels of PLX51107 were evaluated in cancer patients. The MYC PIA assay showed that suppression of MYC appeared at 30 mg/day and peaked at 120 mg/day (Figure 3A). A further increase in dose to 160 mg/day did not lead to enhanced MYC suppression. For each plasma sample that was used to assay for MYC PIA, the plasma concentration of PLX51107 within that sample was meas- ured. When the MYC PIA results are plotted against con- centrations of the drug, the points align with the standard curve (see Figure 2B) of PLX51107 in plasma (Figure 3B). We also plotted all MYC PIA values from the 120 mg/day cohort (Figure 3C), from which we estimate the duration of MYC inhibition to be approximately 6 h at this dose. The mean plasma concentration over the first 6 h is 3.3 μM, which achieves approximately 80% inhibition of MYC in the PIA. An equivalent effect is achieved in regular culture medium by PLX51107 at a concentration of 250 nM.
Even potent FLT3 inhibitors such as gilteritinib and quizartinib are unable to eradicate FLT3-ITD AML blasts on BM stroma as single agents (Figure 4A), which we and others have observed previously.9,10,14,15 Having established that PLX51107 could achieve target inhibition and tumor control in vivo, we next asked if stromal-mediated resist- ance to FLT3 inhibition in vitro could be overcome with the addition of BET inhibition. In previous studies, the con- centration of quizartinib necessary to achieve complete FLT3 inhibition in blasts on BM stroma was established to be 50 nM (in culture medium).24,25 From the above experi- ments, the plasma concentration of PLX51107 in patients (Figure 3B) that resulted in MYC inhibition can be achieved by 250 nM PLX51107 in cell culture medium (Figure 2A). Therefore, primary blast samples from patients with FLT3-ITD AML were co-cultured with mes- enchymal stromal cells obtained from healthy marrow donors in the presence of 50 nM quizartinib, 250 nM PLX51107, or the combination, for 72 h, and then the cells were assayed for viability compared with untreated con-
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