Phase I Study Combining the Aurora Kinase A Inhibitor Alisertib with mFOLFOX in Gastrointestinal Cancer

Abstract

Overexpression and cellular miss-localization of aurora kinase A (AURKA) in gastrointestinal cancers results in chromosomal instability, activation of multiple oncogenic pathways, and inhibition of pro-apoptotic signaling. Inhibition of AURKA causes mitotic delays, severe chromosome congression, and activation of p53/p73 leading to cell death. Our preclinical data showed cooperative activity with the AURKA inhibitor alisertib and platinum agents in cell lines and xenografts, and suggested an optimal treatment window. Therefore, this study was designed to determine the maximum-tolerated dose (MTD) of alisertib in combination with modified FOLFOX (mFOLFOX), as this is a standard platinum-based therapy for gastrointestinal cancers. Standard 3+3 dose escalation was used, where the starting dose of alisertib was 10 mg twice daily (Days 1–3), with leucovorin (400 mg/m²) and oxaliplatin (85 mg/m²) on Day 2 followed by continuous 46-hour 5-FU (2400 mg/m²) infusion on Days 2–4 in 14-day cycles. Fourteen patients with advanced gastrointestinal cancers were enrolled and two doses explored; two patients were not evaluable for dose-limiting toxicity (DLT) and replaced. Two patients experienced DLTs at 20 mg of alisertib (Grade 3 fatigue (n=2); Grade 3 nausea, vomiting, dehydration with hospitalization (n=1)). MTD was 10 mg alisertib with 85 mg/m² oxaliplatin and 2400 mg/m² 5-FU. Most frequent toxicities were nausea (57%), diarrhea, fatigue, neuropathy, and vomiting (43%), and anorexia and anemia (36%); most were Grade 1–2. One patient with colorectal cancer had a partial response of 12 evaluable patients, and four patients had stable disease. Alisertib in combination with mFOLFOX did not demonstrate unexpected side effects, but the regimen was only tolerable at the lowest dose investigated.

INTRODUCTION

The aurora kinase family of serine/threonine kinases has three members, designated aurora kinases A, B, and C [1]; kinases A and B are expressed in many different cell types, whereas the expression of kinase C is restricted to testicular tissue. Aurora kinases localize in the centrosome and play a crucial role in cell division by regulating the segregation of chromatid in cells undergoing mitosis [2]. Specifically, aurora kinase A (AURKA) is located at the centrosome in early S phase, and a fraction associates with the mitotic spindle during cell division [2–4]. Because AURKA is required for cytokinesis, inhibition of AURKA causes spindle pole and chromosome congression defects leading to aneuploidy, which is then followed by cell death [5–8]. Knockdown of AURKA was shown to suppress centrosome maturation [4,9]; in mice, genetic ablation or null mutation of AURKA caused mitotic arrest and embryonic death [10–12].

In addition to its primary effects on the cell cycle, AURKA appears to have a role in oncogenic signaling, including c-MYC, CCND1, and VEGF [13,14]. Several studies have shown that AURKA overexpression directly leads to malignant transformation and subsequently tumor formation [15–17]. Overexpression or amplification of AURKA has been identified in several solid malignancies, including colorectal, gastric, esophageal, liver, and pancreas cancers [18,15,19–23]. Notably, overexpression was observed in multiple aggressive gastrointestinal adenocarcinomas [24,25]. Furthermore, it has been suggested that AURKA is an essential mediator of chemo-resistance in colorectal cancer [26]. Collectively, these findings strongly suggest that targeting AURKA, either alone or combination, in gastrointestinal cancers may represent an attractive treatment option.

Alisertib (MLN8237) is an oral, selective inhibitor of AURKA [27]. In preclinical studies, single agent alisertib showed potent inhibition of AURKA and high antitumor activity [27,28], as well induced abnormal G2/M cell cycle arrest in upper gastrointestinal cancers [29–31]. Combination studies in multiple in vitro and in vivo models demonstrated additive activity or better. Combination treatment with docetaxel enhanced apoptosis and antitumor activity in lymphoma [32] and upper gastrointestinal adenocarcinomas [31]. In B-cell Non-Hodgkin Lymphoma, alisertib administered with vincristine and rituximab resulted in robust cell death [33].

We have previously observed an added benefit of combining alisertib with platinum agents, both cisplatin and oxaliplatin, in multiple human esophageal (FLO-1) and gastric cancer (AGS, Kato-III) cell lines, as well as two esophageal (FLO-1 and OE33) xenograft models [34,35]. Based on our preclinical data, we designed a phase I study to evaluate safety and tolerability of alisertib in combination with modified FOLFOX (mFOLFOX) in patients with gastrointestinal cancers. Additionally, our observations from the in vitro and in vivo studies indicated that alisertib has a delayed apoptotic effect, inducing polyploidy in the first 24 hours followed by cell death in 72–96 hours, suggesting an optimal timing window. Antitumor activity was evaluated as a secondary objective. Exploratory correlatives were performed to evaluate the potential relationship between expression levels of AURKA and c-MYC with tumor response.

MATERIALS AND METHODS

Patient Selection

This investigator-initiated, multiple-institution phase I study (NCT02319018) of alisertib with mFOLFOX was conducted in patients with metastatic or unresectable gastrointestinal cancers where standard therapies did not exist or were no longer effective, or for whom FOLFOX was appropriate. Eligible patients were >18 years of age with an Eastern Cooperative Oncology Group (ECOG) performance status (PS) of 0–1, had adequate organ and marrow function, were able to swallow oral medication, and did not have grade ≥2 peripheral neuropathy. Prior treatment with FOLFOX was allowed.

Study Treatment

Dose escalation (Table 1) began with 10 mg of alisertib twice daily on Days 1–3,with leucovorin and oxaliplatin (85 mg/m²) on Day 2 followed by continuous 5-FU (2400 mg/m²) infusion on Days 2–4 in 14-day cycles. The mFOLFOX regimen did not include the 5-FU bolus as previous study demonstrated that the bolus is responsible for most of the cytopenias and could be removed without compromising efficacy [36]. Dose escalation was planned for groups of three patients until the maximum-tolerated dose (MTD) was established in a standard 3+3 design. Dose limiting toxicity (DLT) was defined as any treatment-related, Grade ≥3 non-hematologic toxicity (except nausea, vomiting, and diarrhea) or Grade ≥4 hematologic toxicity experienced within the first two cycles (28 days). Nausea, vomiting, or diarrhea were dose limiting when Grade 3 toxicity occurred despite optimal use of anti-emetic or anti-diarrheal agents, or lasted longer than 48 hours. A treatment delay of >21 days due to a treatment-related toxicity was also dose limiting. Patients must have received two doses of mFOLFOX and at least 80% of the planned doses of alisertib to be evaluable for DLT, unless missed doses were due to a DLT.

Table 1.

Dose Escalation Schema (standard 3+3 design)

Dose Level	MLN8237 (Days 1–3)	5-FU CI (Total given over Days 2–4)	Oxaliplatin (Day 2)	First two cycle (28 days) Toxicity
Level 1*	10 mg BID	2400 mg/m2	85 mg/m2	6 patients; 0/6 with DLT
Level 2*	20 mg BID	2400 mg/m2	85 mg/m2	6 patients; 2/6 with DLT+
Level 3	30 mg BID	2400 mg/m2	85 mg/m2	Not Evaluated
Level 4	40 mg BID	2400 mg/m2	85 mg/m2	Not Evaluated
Level 5	50 mg BID	2400 mg/m2	85 mg/m2	Not Evaluated

^*Maximum tolerated dose investigation limited to these levels;

⁺Grade 3 fatigue (n=2); Grade 3 nausea, vomiting, dehydration with hospitalization (n=1)

CI: continuous infusion; BID: twice daily

Patients continued on study until unacceptable toxicity, disease progression, patient withdrawal, or specific changes in a patient’s condition that, in the judgment of the investigator, rendered the patient unacceptable for further treatment. Patients were followed for four weeks after removal from the study or until death, whichever occurred first. Patients removed from study due to unacceptable adverse events were followed until resolution or stabilization of the adverse event.

Study Assessments

Toxicity assessments were performed at each cycle and graded according to the NCI Common Toxicity Criteria, Version 4.0. Patients were evaluable for toxicity from the time of their first treatment with alisertib. Patients that were removed from the study during the first four weeks of treatment for reasons other than progressive disease or drug-related adverse events were not considered evaluable for DLT and were replaced; however, these patients were evaluable for toxicity. Disease assessments were performed at baseline and every eight weeks using RECIST v1.1. Only those patients that had measurable disease at baseline, received at least one cycle of therapy, and had their disease re-evaluated were evaluable for response. Patients who were removed during the first two cycles due to progressive disease were also evaluable for response.

Correlative Biomarker Analysis

The primary goal of the correlative studies was to explore the feasibility of performing biomarker assays on human samples in gastrointestinal cancers and guide biomarker development for later phase studies. Consenting patients provided archival tissue. Immunohistochemistry (IHC) for expression levels of AURKA and c-MYC was performed using a rabbit anti-human AURKA polyclonal antibody (Transgenic Inc., Japan) and a rabbit monoclonal [Y69] anti-human c-Myc (Abcam, USA), respectively, following the manufacturers’ recommended protocols.

Statistical Analysis

Descriptive statistics were used to summarize demographics, adverse events, and tumor response. Confidence intervals were estimated using the Wilson method, and survival functions were estimated using the Kaplan-Meier method. Correlations between tumor response and expression levels of AURKA and c-MYC were evaluated.

RESULTS

Patient Characteristics

Between November 2015 and February 2017, 14 patients were enrolled and two dose levels were investigated. Baseline patient characteristics are listed in Table 2. One patient exhibited disease progression prior to completing the DLT observation window and was replaced. Another patient was found to be receiving prohibited medications during the DLT observation window and was replaced. Median age was 59 years (range: 27–80) and 64% were male. Various gastrointestinal malignancies were evaluated: colon (n=4), pancreas (n=4), stomach (n=2), bile duct (n=2), esophagus (n=1), and appendix (n=1). Twelve patients were evaluable for toxicity. Of these 12, two came off study due to unacceptable toxicity while the remaining 10 continued treatment until disease progression.

Table 2.

Patient Baseline Characteristics (n=14)

Gender - No. (%)
Male	9 (64)
Female	5 (36)
Median age - No. (range)	59 (27 − 80)
Race - No. (%)
White	8 (57)
Black	4 (29)
Asian	2 (14)
Ethnicity - No. (%)
Hispanic	1 (7)
Non-hispanic	12 (86)
Not Reported	1 (7)
Primary Site of Disease - No. (%)
Colon	4 (29)
Pancreas	4 (29)
Stomach	2 (14)
Bile Duct	2 (14)
Esophagus	1 (7)
Appendix	1 (7)
PS (ECOG) - No. (%)
0	3 (21)
1	11 (79)

PS: performance status; ECOG: Eastern Cooperative Oncology Group

Treatment and Toxicities

Three patients were enrolled in dose level 1, and zero DLTs were observed. At dose level 2 (20 mg BID alisertib, 85 mg/m² oxaliplatin, and 2400 mg/m² 5-FU), two of three patients experienced DLTs: Grade 3 fatigue (n=2); Grade 3 nausea, vomiting, and dehydration with hospitalization (n=1). Alisertib was de-escalated to 10 mg twice daily (dose level 1) and three additional patients were enrolled; no DLTs observed. Thus, the MTD was 10 mg BID alisertib, 85 mg/m² oxaliplatin, 400 mg/m2 leucovorin and 2400 mg/m² 5-FU.

The most common treatment-related toxicities were nausea (57%), diarrhea (43%), fatigue (43%), peripheral sensory neuropathy (43%), vomiting (43%), anorexia (36%), and anemia (36%); a majority of these were Grade 1–2. Other frequently observed (≥20%) adverse events are listed in Table 3. Eight patients experienced at least one Grade 3 adverse event, with the most common being neutropenia (29%). Treatment-related Grade 4 hypokalemia was reported in one patient. All Grade 3–4 adverse events are listed in Table 3.

Table 3.

Treatment-related adverse events reported in ≥ 20% of patients (n=14)

Adverse Event	All grades	Grade ≥3*
Non-hematological
Nausea	8 (57%)	1 (7%)
Diarrhea	6 (43%)	0
Fatigue	6 (43%)	2 (14%)
Peripheral Sensory Neuropathy	6 (43%)	1 (7%)
Vomiting	6 (43%)	1 (7%)
Anorexia	5 (36%)	0
Elevated Alanine Aminotransferase	4 (29%)	0
Elevated aspartate Aminotransferase	3 (21%)	0
Oral Mucositis	3 (21%)	0
Hematological
Anemia	5 (36%)	1 (7%)
Neutropenia	4 (29%)	4 (29%)
Leukopenia	4 (29%)	0
Lymphocytopenia	3 (21%)	0
Thrombocytopenia	3 (21%)	0

^*Other related grade 3 events reported in one patient each were: dehydration, anaphylaxis, and hypophosphatemia. A treatment-related grade 4 hypokalemia was reported in one patient.

Antitumor Activity

One patient did not have measurable disease at baseline and was not evaluable for response. Of the 12 evaluable patients (Table 4), one patient with colon cancer had a partial response for an 8.3% response rate; this patient had prior FOLFOX in the adjuvant setting. Interestingly, AURKA was amplified in the tumor of this patient. An additional four patients had stable disease for a 42% disease control rate; of these patients, one had adjuvant FOLFOX, one had FOLFOX and CAPOX for the treatment of advanced disease, and the remaining two did not have prior FOLFOX. One patient with esophageal cancer achieved a −28% tumor regression, but unfortunately came off study after four cycles due to excessive toxicity. The median time on treatment was 1.9 months (Figure 1B, Figure 2A; range: 0.5 – 9.2 months); two patients with colon cancer were on study longer than six months. The overall mean progression-free survival (PFS) was 2.3 months (95% confidence interval: 1.4 – 6.2 months).

Table 4.

Best Response According to RECIST for each efficacy evaluable patient

Subject ID No.	Disease Site	Alisertib Dose (BID)	Best Response	Best Percent Change	Reason Off Study	AURKA Status
001	Pancreas	10 mg	PD	−0.7%	Toxicity	No Alteration Detected
002	Bile Duct	10 mg	PD	+12%	Progression	No Alteration Detected
005	Esophagus	20 mg	SD	−28%	Toxicity	Unknown
006	Colon	20 mg	SD	−6.3%	Progression	Unknown
007	Pancreas	20 mg	PD	+24%	Progression	Unknown
008	Colon	20 mg	PR	−37%	Progression	AURKA Amplification
009	Stomach	20 mg	SD	−5.9%	Progression	No Alteration Detected
010	Appendix	20 mg	SD	0.0%	Toxicity	Unknown
011	Pancreas	10 mg	PD	+28%	Progression	Unknown
013	Colon	10 mg	PD	+34%	Progression	Amplification equivocal
014	Colon	10 mg	PD	+24%	Progression	Unknown

RECIST: response evaluation criteria in solid tumors; No.: number; BID: twice daily; PD: progressive disease; SD: stable disease; PR: partial response; AURKA: aurora kinase a

Figure 1. Consolidated Standards of Reporting Trials Diagram.

Study diagram listing number of eligible subjects enrolled onto the study, and numbers of patients in the safety, efficacy, and biomarker correlate analyses.

Figure 2. Antitumor Activity of Alisertib in Combination with mFOLFOX.

(A) Swimmer plot showing time on treatment and duration of stable disease or partial response where applicable. (B) Kaplan-Meier analysis for progression-free survival displayed with 95% CIs.

Correlative Biomarker Analysis

Expression levels of AURKA and c-MYC were analyzed using archival tissue from six patients. Representative IHC sections are shown for two patients in Supplemental Figure 1, showing minimal cytosolic staining of AURKA and c-MYC and varying degree of nuclear staining for both markers. IHC scores, primary site of disease, and best response for the six patients are listed in Supplemental Table 1. A correlation between expression levels of AURKA and c-MYC with tumor response was not observed, possibly due to the small number of tissues analyzed.

DISCUSSION

Gastrointestinal cancers are a leading cause of cancer-related deaths; specifically, in the United States, colorectal cancers are the second and third leading cancer-related deaths in men and women, respectively [37]. Moreover, the number of pancreatic cancer deaths have been on the rise for the past few decades [37]. Esophageal and gastric cancers, although more rare than other gastrointestinal cancers, are also deadly with <10% 5-year survival for patients with metastatic disease [38]. Thus, the need for novel treatment modalities and targeted therapeutics, that are based on the molecular features of these tumors, are needed to improve survival. Our preclinical data showed that inhibiting AURKA in combination with platinum-based chemotherapies significantly decreased cell viability and survival, as well as significantly inhibited xenograft tumor growth in multiple gastrointestinal cancer models compared to either single agent alone, thus, suggesting a potential synergistic treatment combination. Based on these data, a phase I study was designed to assess the safety and tolerability of alisertib in combination with mFOLFOX, as this is an appropriate platinum-based chemotherapy regimen for most gastrointestinal cancers.

In general, alisertib administered with mFOLFOX did not identify any unexpected side effects. The toxicity profile was similar to a phase II study investigating single agent alisertib in gastroesophageal adenocarcinoma [39], with the exception of increased frequencies of leukopenia, nausea, and decreased appetite that is probably attributable to FOLFOX. However, alisertib in combination with FOLFOX was only tolerable at the lowest dose evaluated (i.e., 10 mg BID), which is significantly lower than the recommended phase II dose (RP2D) of single-agent alisertib (50 mg BID) [39–41]. The 5-FU bolus was already removed to reduce the frequency of neutropenias, therefore it was decided not to decrease the dose 5-FU or oxaliplatin in order to increase alisertib as those standard doses have a known benefit in patients with gastrointestinal malignancies.

Of the 12 patients with measureable disease, clinical activity was seen in approximately half (42%). Two patients with colon cancer were on study longer than six months, and one of those patients achieved a partial response. Both patients had previously received FOLFOX in the adjuvant setting. Interestingly, AURKA was amplified in the tumor of the responder. (Unfortunately, the AURKA status was unknown in the other colon cancer patient.) Alisertib in combination with mFOLFOX in the current study resulted in less clinical activity than other combination studies. A phase I study of alisertib with docetaxel demonstrated a 28% response rate in advanced solid tumors [42]. Another phase I study in neuroblastoma observed an overall response rate of 31.8% (50% response rate in patients treated at the MTD) when alisertib was combined with irinotecan and temozolomide [43].

The limited clinical activity observed in this study could be attributed to a number of factors. Variable IC50 values of alisertib have been shown across multiple cancer models; an IC₅₀ of 2 nM was observed in chronic myeloid leukemia [44,45]. Yet in other cancers, IC₅₀ values range from 6.7 nM in cervical (HeLa) cancer cells to as high as 469 nM in the DLD-1 colon cancer cell line [27]. Furthermore, Pitts, et al. demonstrated varying sensitivity to alisertib across multiple colorectal cancer cell lines and xenograft models [46], which might explain the variable clinical activity observed in this study, especially between the patients with colorectal cancer. Another logical reason for the limited clinical activity could be the associated toxicity with the combination regimen. The only tolerable dose of alisertib was 10 mg BID in combination with the standard dose of mFOLFOX, which is significantly lower than the RP2D of single-agent alisertib.

Despite the potential for differences in alisertib sensitivity between patients in this trial combined with the low tolerable dose, two patients were on study longer than six months and one of those patients achieved a partial response. Thus, alisertib administered with oxaliplatin-based regimens could be beneficial in patients with gastrointestinal malignancies, especially in those whose tumors have alterations in AURKA expression or function. However, a phase II study requiring an AURKA alteration or overexpression for inclusion would be needed in order to fully characterize the antitumor activity of this regimen in gastrointestinal cancers.

CONCLUSIONS

This Phase I study was designed to evaluate a novel therapeutic regimen, combining the AURKA inhibitor alisertib with mFOLFOX in patients with advanced gastrointestinal cancers. Although this regimen was safe with manageable side effects, alisertib in combination with standard FOLFOX was only tolerable at the lowest dose evaluated; the MTD was 10 mg BID alisertib administered with 85 mg/m² oxaliplatin and 2400 mg/m² 5-FU. Limited clinical activity was observed; however, one patient whose tumor overexpressed AURKA experienced a partial response, suggesting the need to further explore biomarkers of AURKA inhibition to select patients who may potentially benefit from AURKA inhibitors in gastrointestinal malignancies.