CPX-351 in FLT3-mutated acute myeloid leukemia

CPX-351, a dual-drug liposomal encapsulation of daunorubicin and cytarabine in a 1:5 molar ratio, is approved for the treatment of newly diagnosed therapy-related acute myeloid leukemia (AML) or AML with myelodysplasia-related changes. In a pivotal phase III trial, CPX-351 significantly improved overall survival compared with standard-of-care 7 + 3 chemotherapy (7 days cytarabine; 3 days daunorubicin) in adults aged 60–75 years with newly diagnosed high-risk or secondary AML (median = 9.56 months vs. 5.95 months; hazard ratio = 0.69; 95% confidence interval = 0.52–0.90; p = 0.003). Approximately 30% of patients with newly diagnosed AML have mutations in the FLT3 gene, which may be associated with poor outcomes. Here, we review the current in vitro, clinical, and real-world evidence on the use of CPX-351 in patients with AML and mutations in FLT3. Additionally, we review preliminary data from clinical trials and patient case reports that suggest the combination of CPX-351 with FLT3 inhibitors may represent another treatment option for patients with FLT3 mutation-positive AML.

FLT3 is expressed in most AML blasts and plays a key role in normal hematopoiesis and leukemogenesis (7,8).Approximately 30% of patients with newly diagnosed AML have mutations in the FLT3 gene, most commonly an internal tandem duplication (ITD; ~25% of cases) or point mutations in the tyrosine kinase domain (TKD; 7%-10% of cases) (9).Among patients with t-AML and normal karyotype, the frequencies of FLT3-ITD and FLT3-TKD mutations are 23% and 9%, respectively (10).The FLT3-ITD mutation is associated with poor outcomes in patients with AML, whereas the prognostic impact of FLT3-TKD mutations is unclear (9).Here, we review the preclinical and clinical evidence on the use of CPX-351 in patients with AML and mutations in FLT3.

Ex vivo/in vitro data
Following initial reports of CPX-351 activity compared with conventional drug combinations in phase II trials, the cytotoxic activity of CPX-351 was assessed across risk groups using an ex vivo assay with freshly harvested blasts from 53 patients with AML (11).Primary AML leukemia blasts showed high sensitivity to CPX-351, with 50% growth inhibition concentration (IC 50 ) values ranging from 0.035:0.007mM to 9.77:1.95mM.Responses to CPX-351 were similar across conventional cytogenetic risk groups (per 2010 European LeukemiaNet criteria), including blasts with intermediate II or adverse cytogenetic and molecular abnormalities, and were not correlated with prior response to 7 + 3 treatment.In total, samples from 14 patients with AML were FLT3-ITD-positive, and these blasts were found to be nearly five times more sensitive to CPX-351induced cytotoxicity than FLT3-ITD-negative AML blasts (mean IC 50 values = 0.29:0.058mM and 1.32:0.26mM, respectively; p = 0.047 for difference).In contrast, mutations in NPM1 or CEBPA were found to have no significant impact on CPX-351 treatment responses.FLT3-ITD-positive AML blasts were also observed to have an increased uptake of CPX-351 as compared with FLT3-ITD-negative blasts (Figure 1).Overall, the sensitivity of AML blasts to CPX-351 in this ex vivo assay was consistent with the observed clinical activity in patient populations (11).
The above findings were corroborated by analysis of AML cell lines (MOLM-13, MOLM-14, and ME1), which also showed increased sensitivity to CPX-351 and increased drug uptake in the presence of FLT3-ITD or FLT3-activating mutations, compared with other genetic abnormalities (12).It has been hypothesized that dysregulation of FLT3 signaling may lead to the activation of liposome uptake pathways, resulting in increased sensitivity to CPX-351.Notably, pretreatment of AML cell lines with the FLT3 inhibitor, quizartinib, for 16 h resulted in ~50% of the total cell population exhibiting a decrease in daunorubicin fluorescence, an indicator of drug uptake, suggesting that prolonged FLT3 inhibition may decrease CPX-351 uptake.It follows that alterations in the timing of FLT3 inhibitor treatment may impact effectiveness.Consistent with this, it was found that combining CPX-351 with FLT3 inhibitors (quizartinib and midostaurin) had a synergistic effect when the drugs were used simultaneously or when CPX-351 was scheduled 24 h before the FLT3 inhibitor.However, when FLT3 inhibitors were administered 24 h prior to CPX-351, less synergy was seen, and at certain doses, the effects were antagonistic (12).The authors concluded that these results provide additional evidence that FLT3 activation leads to increased uptake of CPX-351.
A German real-world study of first-line CPX-351 efficacy and safety in adults with newly diagnosed AML-MRC or t-AML (n = 188) included 13 patients with FLT3-ITD mutations (13).The CR/CRi rate was 58% in patients with FLT3-ITD mutations and 48% in patients with wild-type FLT3-ITD (p = 0.45 for difference).The presence of a FLT3-ITD mutation was not found to be a factor impacting OS in univariate analysis (HR = 0.4; 95% CI = 0.2-1.1;p = the CR/CRi rate was 60% (n = 53; there was no statistical difference between the groups; p = 0.72) (14).In patients with FLT3-TKD mutations, the CR/CRi rate was 50% (n = 3), and in patients with nonmutated FLT3-TKD, the CR/CRi rate was 60% (n = 56; p = 0.62  for difference) (14).Another real-world study that supports the data from the phase III study described above is a Canadian multicenter study of 50 patients who received CPX-351 for t-AML and AML-MRC, 10 of whom had mutations in FLT3 (eight ITD and two TKD).Analysis of OS by risk factors showed no differences, including when stratified by the presence of a FLT3-ITD mutation (p = 0.29) (15).Furthermore, in an Italian compassionate use program that recruited patients aged 52-79 years with secondary AML, three of five patients with a FLT3-ITD mutation achieved CR/CRi with CPX-351; the 12month OS was 60% (16).It is important to note that all real-world studies described here had limited numbers of patients with FLT3 mutations; however, these findings support a role for CPX-351 as early-line treatment in patients with FLT3-mutated AML.
Combination treatment with CPX-351 + a FLT3 inhibitor in patients with FLT3-mutated AML The phase Ib V-FAST master study (NCT04075747) was initiated to investigate the recommended phase II dose and the safety and tolerability of CPX-351 when administered in combination with targeted agents in patients with AML who are fit to receive intensive chemotherapy (17,18).Patients with mutated FLT3 were assigned to receive CPX-351 in combination with the FLT3-inhibitor midostaurin.
Preliminary results from the ongoing V-FAST study suggest that the combination of CPX-351 + midostaurin is a feasible treatment strategy for adults with newly diagnosed AML who have an FLT3 mutation (17,18).In total, 23 patients with FLT3-mutated AML received CPX-351 + midostaurin treatment.Of these, 18 (78%) had FLT3-ITD, and six (26%) had FLT3-TKD mutations (one patient had both mutations and was included in both subgroups).Most patients had de novo disease (78% of patients with FLT3-ITD and 100% of patients with FLT3-TKD mutations).The majority of patients achieved CR with CPX-351 + midostaurin, including 14/17 (82.4%) patients with FLT3-ITD mutations, and five of six (83.3%) patients with FLT3-TKD mutations.Among patients with a CR and known measurable residual disease status (as assessed by multicolor flow cytometry at local laboratories), rates of measurable residual disease negativity after induction were 50% for those with FLT3-ITD mutations and 33.3% for those with FLT3-TKD mutations.At the time of analysis, 10 of 18 patients (55.6%) with FLT3-ITD mutations and two of six patients (33.3%) with FLT3-TKD mutation had proceeded to hematopoietic cell transplantation following treatment with CPX-351 + midostaurin.Hematologic recovery times in the V-FAST study were consistent with CPX-351 monotherapy.The overall safety profile was manageable, with serious adverse events reported in 33.3% of patients and no deaths occurring prior to study day 60 (17,18).These findings are supported by case reports from three patients who received CPX-351 + midostaurin for the treatment of FLT3 mutation-positive secondary AML.These were a 75-year-old man with AML-MRC and FLT3-ITD mutation, a 73-year-old woman with t-AML and FLT3-TKD mutation, and a 60-year-old woman with AML-MRC and FLT3-TKD mutation.In each case, combination treatment did not cause any unexpected adverse events and resulted in a CR.Overall, the median time to neutrophil recovery (≥ 500/µL) was 31 days, while the median time to platelet recovery (≥ 50,000/µL) was 32 days.OS in the three treated patients was 17, 4, and 12 months, respectively (19).One of the three patients went on to receive an allogeneic stem-cell transplant and remained in CR post-transplant.

Discussion
This mini-review summarizes the growing evidence supporting a role for CPX-351 in the treatment of AML associated with mutations in FLT3.Furthermore, preliminary data from clinical trials and patient case reports suggest that the combination of CPX-351 + midostaurin may represent an additional treatment option for patients with FLT3 mutation-positive AML.Combination therapies using an induction chemotherapy backbone with targeted agents are increasingly being used in eligible patient populations for the treatment of AML (20).The benefit of combining standard chemotherapy with midostaurin in patients with AML and FLT3 mutations has been demonstrated previously.In the randomized phase III RATIFY study (NCT00651261), the addition of midostaurin to chemotherapy significantly improved OS and event-free survival in adults with newly diagnosed AML and an FLT3 mutation compared with placebo (21).The combination of CPX-351 + FLT3 inhibitors for AML will be further investigated in the phase I/II NCT04128748 study (CPX-351 + quizartinib), the phase I/II NCT04982354 study (CPX-351 + midostaurin), and the phase III NCT04293562 study (CPX-351 + gilteritinib), which are currently recruiting patients.Interestingly, preclinical data have suggested a synergistic effect with combination treatment when FLT3 inhibitors were administered after CPX-351 (12).While the use of CPX-351 as a combination therapy with FLT3 inhibitors continues to be evaluated in clinical trials, the pivotal CPX-351 clinical trial and real-world studies have demonstrated that CPX-351 monotherapy may be of benefit in AML patients with FLT3 mutations; no significant differences in CPX-351 treatment outcomes were observed between patients with versus without an FLT3 mutation.
A caveat to the findings described in this review is the relatively small number of patients included in each analysis; further validation of these data is warranted in larger studies.In addition, the data described in this article also do not consider the allelic ratio of FLT3-ITD mutations, which is known to have an impact on disease prognosis (7).It remains to be determined whether FLT3-ITD high or FLT3-ITD low genotypes show differential responses to CPX-351 treatment.However, using allelic ratio testing in clinical practice presents challenges as it is often unavailable to treating physicians, and there is a lack of validated and standardized methods between laboratories.Furthermore, FLT3-ITD allelic ratios are no longer part of the European LeukemiaNet risk classification at initial diagnosis for AML (22).
Also worthy of consideration and future study are the mechanisms of resistance to CPX-351 treatment, such as TP53 mutation (23), and how these may specifically impact patients with FLT3 mutations.
In summary, the findings reviewed in this article highlight the potential for CPX-351 use in the treatment of patients with AML and mutations in FLT3.Combination approaches with CPX-351 and FLT3 inhibitors are supported by preclinical data and initial findings from clinical studies.

FIGURE 1
FIGURE 1 Correlation of CPX-351 cytotoxicity with cellular uptake in AML blasts.Patient blasts (n = 12) were exposed to graded concentrations of CPX-351 for 24 h, and the number of viable cells at 3 days was used to calculate IC 50 values for each sample.CPX-351 uptake was assessed by analysis of daunorubicin fluorescence uptake using flow cytometry; the ratio of mean fluorescence intensity (MFI) in treated vs. untreated cells was calculated and plotted against CPX-351 IC 50 .The correlation coefficient between IC 50 and MFI was 0.703.Figure reproduced with permission from Gordon et al. 2017 (11).

TABLE 1
Summary of published clinical study data and real-world evidence for use of CPX-351 monotherapy in patients with FLT3-ITD/TKD mutations.