Purpose:

Myelofibrosis is characterized by bone marrow fibrosis, atypical megakaryocytes, splenomegaly, constitutional symptoms, thrombotic and hemorrhagic complications, and a risk of evolution to acute leukemia. The JAK kinase inhibitor ruxolitinib provides therapeutic benefit, but the effects are limited. The purpose of this study was to determine whether targeting AURKA, which has been shown to increase maturation of atypical megakaryocytes, has potential benefit for patients with myelofibrosis.

Patients and Methods:

Twenty-four patients with myelofibrosis were enrolled in a phase I study at three centers. The objective of the study was to evaluate the safety and preliminary efficacy of alisertib. Correlative studies involved assessment of the effect of alisertib on the megakaryocyte lineage, allele burden, and fibrosis.

Results:

In addition to being well tolerated, alisertib reduced splenomegaly and symptom burden in 29% and 32% of patients, respectively, despite not consistently reducing the degree of inflammatory cytokines. Moreover, alisertib normalized megakaryocytes and reduced fibrosis in 5 of 7 patients for whom sequential marrows were available. Alisertib also decreased the mutant allele burden in a subset of patients.

Conclusions:

Given the limitations of ruxolitinib, novel therapies are needed for myelofibrosis. In this study, alisertib provided clinical benefit and exhibited the expected on-target effect on the megakaryocyte lineage, resulting in normalization of these cells and reduced fibrosis in the majority of patients for which sequential marrows were available. Thus, AURKA inhibition should be further developed as a therapeutic option in myelofibrosis.

See related commentary by Piszczatowski and Steidl, p. 4868

This article is featured in Highlights of This Issue, p. 4863

Translational Relevance

The bone marrow of patients with myelofibrosis is characterized by accumulation of atypical megakaryocytes that directly contribute to the disease. Here we demonstrate that the AURKA inhibitor alisertib has an on-target ability to force differentiation of these megakaryocytes and that this is associated with clinical benefit in patients with myelofibrosis.

Myelofibrosis is the most aggressive subtype of the Philadelphia chromosome–negative myeloproliferative neoplasms (MPN), which also includes essential thrombocythemia and polycythemia vera. The vast majority of cases of the MPNs are caused by driver mutations in JAK2, MPL, or Calreticulin (CALR) that lead to hyperactive JAK/STAT signaling (1). Although the FDA-approved therapy ruxolitinib alleviates symptoms and improves quality of life in a subset of patients, responses are limited by toxicity or disease progression (2, 3). Moreover, ruxolitinib fails to target the malignant clone or appreciably reduce the degree of fibrosis (4, 5). Therefore, we investigated alternative therapies that may reduce fibrosis and provide additional therapeutic benefit.

A major feature of myelofibrosis is the presence of atypical, clustered megakaryocytes, which fail to express the essential hematopoietic transcription factor GATA1 (6, 7). A number of studies have led to the hypothesis that these atypical megakaryocytes are drivers of bone marrow fibrosis and potentially other features of the disease (8–10). Nevertheless, direct proof for a fibrosis-inducing role of megakaryocytes in myelofibrosis is lacking. We previously reported that AURKA inhibition leads to polyploidization, partial differentiation, and subsequent apoptosis of malignant megakaryocytes in animal models of myelofibrosis and acute megakaryoblastic leukemia, as well as in primary cells from patients (11, 12). In particular the highly selective AURKA inhibitor alisertib demonstrated potent antitumor effect in both the JAK2V617F and MPLW515L models of myelofibrosis. In addition to eradicating the atypical megakaryocytes, alisertib normalized blood counts and reversed bone marrow fibrosis. Given the limitations in ruxolitinib, which include the development of dose-limiting cytopenias, a modest antifibrotic effect, a high discontinuation rate, and potential for opportunistic infections (2, 13), there is an urgent medical need for new treatments (14). To assess the clinical activity of alisertib in myelofibrosis, we performed an investigator initiated pilot study of alisertib in patients with myelofibrosis (ClinicalTrials.gov Identifier NCT02530619).

Patients

Included patients had a confirmed diagnosis of primary myelofibrosis, post-thrombocythemia/myelofibrosis, or post-polycythemia vera/myelofibrosis; were intermediate-1 risk or beyond by the Dynamic International Prognostic Scoring System (DIPSS); and were in need of treatment, intolerant or refractory to ruxolitinib (or other investigational JAK inhibitors), or unlikely to benefit from ruxolitinib. Patients had an estimated life expectancy of 6 months or greater, an Eastern Cooperative Oncology Group status 0–2, and adequate bone marrow function, including an absolute neutrophil count ≥1,000/mm3 and platelets ≥50,000/mm3. Adequate liver and kidney function were also required, defined as direct bilirubin ≤1.5 × upper limit of normal (ULN), ALT/AST ≤2.5 × ULN, and creatinine <1.5 × ULN, or calculated creatinine clearance > 30 mL/minute. Patients with class III or IV heart failure or myocardial infarction within 6 months were not eligible. The study was approved by the institutional review boards at Northwestern University (Chicago, IL), the Mayo Clinic (Rochester, MN), and the University of Miami (Miami, FL), was conducted in accordance with the U.S. Common Rule, and all patients signed informed consent.

The starting dose for all patients was 50 mg twice a day, administered orally on days 1–7 of each cycle (1 cycle = 21 days), based on the maximum tolerated dose from previous studies in other patient populations. Patients could continue to receive cycles of alisertib until progression of disease or unacceptable toxicity.

The primary objective of this pilot study was to determine the safety profile of alisertib. Adverse events (AE) were defined according to the NCI's Common Terminology Criteria for Adverse Events Version 4.0. The number, frequency, and severity of AEs were recorded every cycle and all patients who received at least one dose of alisertib were considered evaluable for this endpoint. The secondary objective was to determine the preliminary efficacy of alisertib. Responses were categorized according to the revised/modified International Working Group for Myelofibrosis Research and Treatment (IWG-MRT) Response Criteria (15). Patients who had measurable disease at baseline, receive at least 1 cycle of treatment, and had at least one postbaseline disease assessment were considered evaluable for this endpoint. Spleen reduction was assessed by palpation once per cycle, and the Myeloproliferative Neoplasm Symptom Assessment Form (MPN-SAF) was administered once per cycle to follow symptom response.

Correlative methods

Blood samples processing, CD34+ cell culture, and megakaryocyte differentiation.

Peripheral blood (20 mL) was collected from patients at the beginning and the end of each treatment cycle and processed within 24 hours postcollection. Peripheral blood (1 mL) was used to collect the serum fraction by two centrifugations at 1,000 × g for 20 minutes and 2, 000 × g for 10 minutes. The mononuclear cell fraction of peripheral blood was isolated through Ficoll (GE Healthcare) by centrifugation at 400 × g for 30 minutes at room temperature. CD34+ progenitor cells were enriched through positive selection from peripheral blood using the Miltenyi Biotech MACS system. The CD34-negative fraction from the selection was saved and frozen for allele burden analysis. Viable CD34+ cells were counted using trypan blue exclusion. CD34+ cells were then cultured for 5 days in StemSpan Media (Stemcell Technologies) supplemented with 50 ng/mL human IL6, 100 ng/mL human FLT-3, 50 ng/mL human SCF, and 30 μg/mL human low-density lipoprotein (Stemcell Technologies). Megakaryocyte differentiation was performed by culturing CD34+ cells in 1:1 RPMI1640 media and StemSpan media supplemented with 50 ng/mL human thrombopoietin (Stemcell Technologies) for 8 days.

In vitro treatment of megakaryocyte differentiating cultures with ruxolitinib or alisertib.

For in vitro drug treatments, CD34+ cells at the end of the 5 day expansion phase were cultured in 10% FBS in RPMI1640 media supplemented with 50 ng/mL human thrombopoietin in the presence of various concentrations of either ruxolitinib or alisertib for 72 hours. After 72 hours, megakaryocyte differentiation and apoptosis were assessed by Hoechst 33342 (Invitrogen) staining for 1 hour at 37°C with 5% CO2 for DNA content analysis and Annexin V staining for apoptosis on CD41+CD42+ megakaryocytes. Flow cytometric analysis was performed on an LSRII flow cytometer (BD Biosciences) and postacquisition analysis performed with FlowJo.

Luminex cytokine array and TGF-β ELISA.

To assess multiple cytokines in a single sample, a ProcartaPlex immunoassay was used to assess 45 different cytokines in the serum of patients pre- and posttherapy. Peripheral blood–derived serum samples were thawed on ice and centrifuged at 10,000 × g for 5 minutes. Magnetic beads for the multiplex assay were prepared according to the manufacturer's instructions and added to a 96-well plate. Samples and blanks were added to the beads and incubated overnight at 4°C. After two plate washes, detection antibody was added to the samples and incubated for 30 minutes at room temperature. The plate was washed again two times, incubated with Streptavidin-PE for 30 minutes at room temperature, and washed two times. Beads were resuspended in 120 μL of reading buffer and reading performed on a Luminex 200. To assess active TGF-β1 levels 1N HCl was added to the serum to activate latent TGF-β1 form and then the acidified sample was neutralized with 1.2 N NaOH/0.5 mol/L HEPES. The sample was diluted 20-fold and assayed with a Quantikine ELISA Kit (R&D Systems) according to the manufacturer's instructions. The plate was read using a microplate reader set at 450 nm. A standard curve was generated by four parameter logistic curve fit and unknown values interpolated from the standard curve.

GATA1 intracellular staining on alisertib-treated SET2 cells.

SET2 cells, which were purchased from DSMZ in 2017, validated by short tandem repeat testing and determined to be Mycoplasma free by PCR in January 2018, were cultured for less than 3 months after thawing. Cells were treated with either DMSO or 1 μmol/L alisertib for 72 hours. After treatment, cells were stained for 1 hour with Hoechst 33342 for DNA content analysis and then fixed in 1.5% PFA for 15 minutes at room temperature. After fixation, cells were permeabilized with ice-cold methanol for 2 hours at 4°C and then stained with a PE-conjugated anti-GATA1 antibody (R&D, catalog no. IC1779P) for 30 minutes at room temperature in the dark for flow cytometric analysis. Samples were analyzed on an LSRII using FlowJo software.

qRT-PCR.

qRT-PCR analysis was carried out on SET2 cells treated for 72 hours with 300 nmol/L alisertib for a total of three biological replicates. Briefly, total RNA was extracted from cells with a Direct-zol RNA MiniPrep Kit (Zymo Research). Five-hundred nanogram of total RNA were reverse transcribed into cDNA with Superscript IV First-Strand Synthesis System (Thermo Fisher Scientific). qPCR reactions were performed with SYBR Green chemistry method using PerfeCta SYBR Green FastMix (Quantabio) and 2 μL of cDNA template. Amplification of the reactions was performed on a StepOnePlus real-time PCR system. Calculation of fold changes relative to the untreated group was performed with the 2(−ΔΔCt) method. Primer sequences are available upon request.

GATA1 IHC.

GATA1 staining of sections of bone marrow biopsies was performed using an anti-GATA1 antibody (Cell Signaling Technology, catalog no. 3535) as described previously (7). The percentage of GATA1-positive megakaryocytes represents the number of positively stained cells relative to the total number of megakaryocytes. A minimum of 100 megakaryocytes were counted, with the exception of 42-10, where limited tissue was available. Images were obtained with an Olympus BX41 microscope fitted with a Jenoptik ProgRes Speed XT camera.

Statistical analysis.

Data are presented as frequencies and percentages or as medians and ranges. The alisertib effect on mean fluorescence intensity was analyzed using the unpaired two-sided Student t test.

Seventeen patients with primary myelofibrosis, 4 with post-thrombocythemia/myelofibrosis, and 3 with post-polycythemia vera/myelofibrosis were enrolled (Table 1). The median age was 72 years with 67% males. A total of 33% of patients were DIPSS intermediate-1 risk, 46% were intermediate-2 risk, and the remainder were high risk. Fifteen patients (63%) had prior exposure to JAK inhibitor therapies; 9 of which experienced disease progression, 2 were intolerant, and the rest did not respond. Nine patients (37%) were JAK inhibitor naïve due to transfusion-dependent anemia (n = 6), absence of splenomegaly (n = 2), and physician preference in a patient with high risk genotype. Driver mutational status was as follows: 58% JAK2V617F, 29% CALR, and 13% MPL mutated with the details of associated mutations provided in Supplementary Table S1. Of note, 10 of 15 patients (67%) evaluated, harbored one or more high molecular risk mutations. At study entry, 58% of patients demonstrated palpable splenomegaly ≥5 cm below the left costal margin and 54% were red cell transfusion dependent. The latter included those with an ongoing need for and history of red cell transfusions for symptomatic myelofibrosis–associated anemia and not merely an isolated instance of red cell transfusion. The median MPN-SAF total symptom score (TSS) was 31, with a range of 2 through 70 (Table 1).

Table 1.

Baseline patient characteristics

CharacteristicsAll patients (n = 24)
Median age (range) 72 (48–80) 
Male, n (%) 16 (67) 
Female, n (%) 8 (33) 
Race, n (%) 
 Caucasian 22 (92) 
 Other 2 (8) 
Myelofibrosis classification, n (%) 
 Primary 17 (71) 
 Post-thrombocythemia/myelofibrosis 4 (17) 
 Post-polycythemia vera/myelofibrosis 3 (13) 
DIPSS risk classification, n (%) 
 Intermediate-1 8 (33) 
 Intermediate-2 11 (46) 
 High 5 (21) 
Median MPN-SAF score (range) 31 (2–70) 
Palpable splenomegaly, n (%) 14 (58) 
Transfusion dependence 13 (54) 
Median disease duration in years (range) 2.8 (0.08–35.5) 
Median white blood cells (range; × 1097.45 (1.50–91.00) 
Median hemoglobin (range; g/dL) 9.45 (6.10–14.80) 
Median platelets (range; ×109177 (74–1,431) 
Prior use of JAK inhibitor, n (%) 15 (63) 
Mutation type, n (%) 
JAK2 14 (58) 
CALR 7 (29) 
MPL 3 (13) 
CharacteristicsAll patients (n = 24)
Median age (range) 72 (48–80) 
Male, n (%) 16 (67) 
Female, n (%) 8 (33) 
Race, n (%) 
 Caucasian 22 (92) 
 Other 2 (8) 
Myelofibrosis classification, n (%) 
 Primary 17 (71) 
 Post-thrombocythemia/myelofibrosis 4 (17) 
 Post-polycythemia vera/myelofibrosis 3 (13) 
DIPSS risk classification, n (%) 
 Intermediate-1 8 (33) 
 Intermediate-2 11 (46) 
 High 5 (21) 
Median MPN-SAF score (range) 31 (2–70) 
Palpable splenomegaly, n (%) 14 (58) 
Transfusion dependence 13 (54) 
Median disease duration in years (range) 2.8 (0.08–35.5) 
Median white blood cells (range; × 1097.45 (1.50–91.00) 
Median hemoglobin (range; g/dL) 9.45 (6.10–14.80) 
Median platelets (range; ×109177 (74–1,431) 
Prior use of JAK inhibitor, n (%) 15 (63) 
Mutation type, n (%) 
JAK2 14 (58) 
CALR 7 (29) 
MPL 3 (13) 

Patients received a median of 7.5 cycles (range: 1–29 cycles) of therapy. The 7 patients presently on study have received a median of 23 cycles (range: 8–29 cycles). Reasons for treatment discontinuation included progressive disease (n = 5) or lack of response (n = 6) in 11 (46%) patients after a median of 7 cycles (range: 2–19 cycles), toxicity in 4 (17%) patients after a median of 7 cycles (range: 1–29 cycles), and refusal of further therapy in 2 (8%) patients. Of the 5 patients with progressive disease, 2 transformed to acute leukemia with survival of 3 and 6 months, respectively. The remaining 3 patients experienced worsening splenomegaly, or anemia of which 2 deaths occurred, 1 on study, and another 5 months after therapy. As expected, the most common treatment-emergent grade 3 or grade 4 AE included neutropenia (21% and 21%, respectively), thrombocytopenia (21% and 8%), and anemia (21% and 0), with 4% of patients each experiencing, vertigo, diarrhea, elevated alanine aminotransferase, and elevated creatinine (Table 2). Of note, there was only one instance of febrile neutropenia requiring hospitalization. Additional treatment-related, nonhematologic grade 1 or 2 AEs occurring in >10% of patients included: diarrhea in 7 (5, grade 1; 1, grade 2; and 1, grade 3); nausea in 7 (6, grade 1 and 1, grade 2); vomiting in 4 (all grade 1), and mucositis in 4 (all grade 1). Seven patients experienced fatigue (2, grade 1 and 5, grade 2), 6 experienced dizziness (all grade 1), and 13 experienced alopecia (11, grade 1 and 2, grade 2). Four serious AEs were reported, including diarrhea (grade 3, 1 patient), febrile neutropenia (grade 3, twice in the same patient), and cellulitis (grade 2, 1 patient). There were 3 deaths during participation in the study, all due to progressive disease and were unrelated to therapy. A list of all treatment-related AEs is included in the Supplementary Table S2.

Table 2.

Drug-related grade 3 or 4 AEs, n (%)

Grade 3Grade 4
Hematologic AEs 
 Neutropenia 5 (21) 5 (21) 
 Febrile neutropenia 1 (4) 
 Lymphocytopenia 9 (38) 
 Thrombocytopenia 5 (21) 2 (8) 
 Anemia 5 (21) 
Nonhematologic AEs 
 Vertigo 1 (4) 
 Diarrhea 1 (4) 
 Alanine aminotransferase increased 1 (4) 
 Creatinine increased 1 (4) 
Grade 3Grade 4
Hematologic AEs 
 Neutropenia 5 (21) 5 (21) 
 Febrile neutropenia 1 (4) 
 Lymphocytopenia 9 (38) 
 Thrombocytopenia 5 (21) 2 (8) 
 Anemia 5 (21) 
Nonhematologic AEs 
 Vertigo 1 (4) 
 Diarrhea 1 (4) 
 Alanine aminotransferase increased 1 (4) 
 Creatinine increased 1 (4) 

Twenty-two patients received at least 1 cycle of therapy, of which 4 of 14 patients (29%) with baseline palpable splenomegaly ≥5 cm achieved a spleen response, defined as ≥50% reduction for at least 12 weeks (Fig. 1A), 2 of 19 patients (11%) an anemia response with both becoming transfusion-independent, and 7 of 22 patients (32%) experienced a symptom response with a sustained ≥50% reduction in the MPN-SAF TSS (Table 3). Per study protocol, response assessment was according to the revised/modified IWG-MRT Response Criteria (15) and formally performed after a minimum of 6 cycles of therapy. Among 13 such patients who had received a minimum of 6 cycles of therapy, spleen responses were observed in 4 of 7 (57%) patients, 1 of 5 (20%) achieved transfusion independence, and 6 of 13 (46%) achieved symptom response (Fig. 1B and C; Table 3).

Figure 1.

Clinical responses to alisertib. A, Waterfall plot depicting the best percentage change in spleen size for all patients on study with a palpable spleen. B, Waterfall plot depicting the percentage change in spleen size from baseline to cycle 6 for the 7 patients with a palpable spleen who remained on study for at least 6 cycles. C, Waterfall plot depicting the percentage change in MPN-SAF TSS from baseline to cycle 6. The 13 patients who remained on study for at least 6 cycles are shown.

Figure 1.

Clinical responses to alisertib. A, Waterfall plot depicting the best percentage change in spleen size for all patients on study with a palpable spleen. B, Waterfall plot depicting the percentage change in spleen size from baseline to cycle 6 for the 7 patients with a palpable spleen who remained on study for at least 6 cycles. C, Waterfall plot depicting the percentage change in MPN-SAF TSS from baseline to cycle 6. The 13 patients who remained on study for at least 6 cycles are shown.

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Table 3.

Clinical responses

Number evaluableNumber of responders (%)
Type of response 
 Symptom response 22 7 (32) 
 Spleen response 14 4 (29) 
 Anemia response 19 2 (11) 
Response in 13 patients with a minimum of 6 cycles 
 Symptom response 13 6 (46) 
 Spleen response 4 (57) 
 Anemia response 1 (20) 
Biomarker response 
 Fibrosis response (one grade reduction) 5 (71) 
 Improvement in GATA1 staining 6 (86) 
Number evaluableNumber of responders (%)
Type of response 
 Symptom response 22 7 (32) 
 Spleen response 14 4 (29) 
 Anemia response 19 2 (11) 
Response in 13 patients with a minimum of 6 cycles 
 Symptom response 13 6 (46) 
 Spleen response 4 (57) 
 Anemia response 1 (20) 
Biomarker response 
 Fibrosis response (one grade reduction) 5 (71) 
 Improvement in GATA1 staining 6 (86) 

Four of 9 (44%) ASXL1-mutated patients were among the responders; 3 patients with spleen and symptom response, and a patient with anemia and symptom response. Notably 2 of the 3 patients with spleen and symptom response had received a prior JAK inhibitor. All patients presenting with leukocytosis (n = 4) and thrombocytosis (n = 2) had resolution with therapy. Of the 7 patients currently on study, 4 patients (57%) continue to demonstrate symptom response, 2 patients (29%) with both spleen and symptom response, and another patient (14%) with sustained anemia response. A synopsis of the correlations between molecular status, prior exposure to JAK inhibitors with response and AEs is provided in Supplementary Table S1. We observed clinical improvement in 3 of the 15 (20%) of patients who failed a JAK inhibitor, and in 4 of the 9 (44%) who were JAK inhibitor naïve. Although this suggests that the outcomes are better in the JAK inhibitor–naïve group, the numbers are too small to draw a definitive conclusion.

We performed a number of correlative studies to assess the impact of alisertib on the disease, focusing initially on its effect on the megakaryocyte lineage. First, we treated primary CD34+ cells from patients in vitro with either ruxolitinib or alisertib and assayed the effect on megakaryopoiesis in vitro (Fig. 2). Alisertib robustly induced polyploidization and apoptosis in a dose-dependent manner in five of five specimens tested. In contrast, the 5 patient samples showed little, if any response (in terms of apoptosis or polyploidization), to ruxolitinib. Of note, 3 of these patients (05-01-14, 05-01-18, and 01-20) had failed prior JAK inhibitor therapy. These results demonstrate that alisertib uniquely affects the megakaryocyte lineage and is active in the setting of JAK inhibitor failure.

Figure 2.

Alisertib but not ruxolitinib enhanced polyploidization and apoptosis of patient-derived megakaryocytes ex vivo. A, Peripheral blood CD34+ cells were cultured in vitro to generate megakaryocytes. hrs, hours; PB, peripheral blood; TPO, thrombopoietin. B, The extent of polyploidy and apoptosis for 5 patient specimens in the presence of increasing doses of alisertib (MLN) versus ruxolitinib (RUX) are shown. Data for 5 individual patients are shown.

Figure 2.

Alisertib but not ruxolitinib enhanced polyploidization and apoptosis of patient-derived megakaryocytes ex vivo. A, Peripheral blood CD34+ cells were cultured in vitro to generate megakaryocytes. hrs, hours; PB, peripheral blood; TPO, thrombopoietin. B, The extent of polyploidy and apoptosis for 5 patient specimens in the presence of increasing doses of alisertib (MLN) versus ruxolitinib (RUX) are shown. Data for 5 individual patients are shown.

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JAK inhibitors have the ability to induce a rapid reduction in inflammatory cytokines, which is likely linked to reduction in splenomegaly (4). To determine whether AURKA inhibition led to a similar cytokine response, we performed Luminex cytokine profiling with 9 paired alisertib-treated patient samples. We failed to see a consistent response, with 2 patients showing an overall decline in the inflammatory cytokines, 3 with a mixed response, and 4 with an overall increase in cytokine levels (Supplementary Fig. S1). In contrast, TGF-β levels did not rise in patients with therapy, and there were several patients with as much as a 50% reduction (Supplementary Fig. S2). Of the 4 patients who demonstrated a spleen response, the cytokine data revealed that there was no correlation between the spleen response and changes in cytokine levels. Although the numbers are small, this observation suggests that alisertib reduces spleen size through a mechanism distinct from ruxolitinib.

We recently reported that the cause of impaired megakaryopoiesis in myelofibrosis is the reduced expression of the hematopoietic transcription factor GATA1 (7). Indeed, atypical megakaryocytes in the bone marrow of patients with MPN and animal models with JAK2, CALR, or MPL mutations fail to stain for GATA1 (6, 7). To investigate whether alisertib, as part of its ability to promote megakaryocyte maturation and polyploidization, could upregulate GATA1 protein levels, we treated the SET2 post-MPN acute myeloid leukemia (AML) cell line with alisertib. By intracellular flow cytometry, we observed robust increase in GATA1 expression in parallel with a marked increase in the ploidy state (Supplementary Fig. S3). To further assess the effect of alisertib on megakaryocytes, we performed qRT-PCR on samples collected from cells treated with DMSO or alisertib for 72 hours. We found that the alisertib-induced differentiation was accompanied by upregulation of megakaryocyte markers CD41b, PF4, VWF, and β1-tubulin (Supplementary Fig. S4). Of note, alisertib did not increase expression of either GATA1 or NFE2, suggesting that the upregulation of GATA1 protein and induction of differentiation occurs in part at the posttranscriptional level.

Next, we compared the intensity of GATA1 staining in sequential bone marrow biopsies from 7 patients at baseline and after a minimum of 5 cycles. We observed a striking increase in GATA1 staining in 6 of 7 cases (86%) examined (Fig. 3; Table 3; Supplementary Fig. S5; Supplementary Table S3). Of note, the GATA1-positive cells included both erythroid cells and megakaryocytes, with an increased proportion of GATA1-positive megakaryocytes in all 6 cases (Supplementary Table S3). Importantly, this increase in staining was accompanied by a normalization in the morphology of this lineage, with restoration of multilobed nuclei and the absence of clustering. The 1 patient whose marrow did not show GATA1 improvement (01-02) entered the study as a patient with post-AML myelofibrosis and who progressed to AML after nearly 1 year of therapy; of note, we observed diffuse GATA1 staining with the blasts at the AML stage.

Figure 3.

Alisertib restored GATA1 staining to megakaryocytes and reduced bone marrow fibrosis. Images of bone marrow biopsies stained with hematoxylin and eosin (H&E), an anti-GATA1 antibody, or for reticulin from patients 42–01 (A) and 01-03 (B) pre- and during therapy. Original magnification, 400×.

Figure 3.

Alisertib restored GATA1 staining to megakaryocytes and reduced bone marrow fibrosis. Images of bone marrow biopsies stained with hematoxylin and eosin (H&E), an anti-GATA1 antibody, or for reticulin from patients 42–01 (A) and 01-03 (B) pre- and during therapy. Original magnification, 400×.

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In addition to the restoration of GATA1 staining and megakaryocyte morphology, we observed a one grade reduction in bone marrow fibrosis in five of seven (71%) paired samples, with one of the nonresponders (01-02) being the one who progressed to AML (Fig. 3; Tables 3; Supplementary Fig. S5; Supplementary Table S3). This reduction in fibrosis was accompanied by sustained responses to the drug. For example, patients 42-01, 01-03, and 42-10 maintained a symptom and spleen response for more than 12 weeks, whereas 01-02 and 05-01-09 exhibited a symptom and anemia response or a symptom response for more than 12 weeks, respectively.

Finally, we compared the mutant allele burden in eight paired baseline and cycle 5 or 6 samples by NGS. We observed decreases in the JAK2, CALR, or MPL allele burdens in 4 of 8 patients examined for whom paired samples were available (Fig. 4). Of note, although the other 4 did not show a decline, the mutant allele burden remained stable. The patients who exhibited the reduction in allele burden at cycle 6 also tended to be those who remained on study longer (01-02, 19 cycles; 05-01-05, 26 cycles; 05-01-09, 26 cycles; and 01-12, 7 cycles).

Figure 4.

Alisertib reduced or stabilized the mutant allele burden in patients on study. Measurement of the variant allele frequencies (VAF) of the driver mutations (MPL, JAK2, or CALR) as well as other genes associated with myeloid malignancies in patients at cycle 1 (T1) and cycle 5 or 6 (T2). Each box represents an individual patient.

Figure 4.

Alisertib reduced or stabilized the mutant allele burden in patients on study. Measurement of the variant allele frequencies (VAF) of the driver mutations (MPL, JAK2, or CALR) as well as other genes associated with myeloid malignancies in patients at cycle 1 (T1) and cycle 5 or 6 (T2). Each box represents an individual patient.

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Here we report the findings of a phase I study that evaluated the activity of a novel therapeutic modality, megakaryocyte differentiation therapy, in the clinic. Although we developed this class of compounds initially to target malignant megakaryocytes in acute megakaryoblastic leukemia, we discovered that these compounds also induced maturation of the atypical megakaryocytes that characterize myelofibrosis (12). Indeed, our preclinical studies revealed that AURKA inhibition potently induced polyploidization, features of maturation, and subsequent apoptosis of megakaryocytes from MPN mouse models and patient samples. Our studies also showed that alisertib suppressed the myeloproliferative and fibrotic phenotypes caused by expression of activated alleles of JAK2 or MPL in animal models of the MPNs.

Our clinical study revealed that alisertib is overall safe and well tolerated, with 7 patients remaining on study of which 3 patients have been on therapy for more than 1.7 years. With respect to clinical benefit, alisertib demonstrated the ability to reduce spleen size and symptom burden both in JAK inhibitor–treated and high-molecular risk patients, two criteria that led to the approval of ruxolitinib for treatment of myelofibrosis.

We evaluated the activity of alisertib in a number of correlative studies. Of note, alisertib improved the morphology and GATA1 staining of the megakaryocyte lineage. Furthermore, unlike ruxolitinib, alisertib therapy was not associated with consistent changes in peripheral blood cytokines, including TGF-β, which has been implicated in driving bone marrow fibrosis. These results indicate that alisertib is not acting as an anti-inflammatory but rather through a novel mechanism targeting megakaryocytes and possibly other lineages as well. Moreover, the finding that patient 01-02 showed clinical improvement despite the lack of improved GATA1 staining suggests that alisertib has antitumor properties that are independent of the megakaryocyte differentiation effect. This effect of alisertib on other cells begs the question of whether the reduction in fibrosis is only due to normalization of megakaryocytes. We cannot exclude the possibility that alisertib affects other lineages, such as osteoblasts, which have been implicated in driving fibrosis in myeloproliferative disorders (16).

With respect to the mechanism by which alisertib leads to increased GATA1 expression in patients, we envision three scenarios. First, it is possible that AURKA inhibits the activity of megakaryocyte differentiation transcription factors, such as NFE2, by direct phosphorylation and that AURKA inhibition then enables this factor to promote maturation, which would be expected to be accompanied by increased GATA1 expression. Second, GATA1 expression may be elevated as a consequence of the increased degree of polyploidization, that is, increased transcription concomitant with increased number of GATA1 alleles. Our gene expression analysis after alisertib treatment (Supplementary Fig. S4) revealed that GATA1 mRNA is not significantly upregulated, suggesting that the increased protein occurs at the posttranscriptional level, a finding that is consistent with our report that there is a defect in GATA1 translation in myelofibrosis megakaryocytes (7). Our data further showed that NFE2 levels are also not increased, consistent with the possibility that its activity may be regulated by AURKA. A third possibility is that the increased proportion of GATA1-positive megakaryocytes seen with treatment reflects an increase in nonmalignant hematopoiesis. However, because alisertib increased GATA1 expression in SET2 cells in culture and did not reduce the mutant allele burden in all cases, the former two possibilities are more likely.

The results of this phase I study are significant in a number of respects. First, we demonstrate that alisertib is safe and well tolerated in patients with myelofibrosis with prolonged administration up to 1.7 years and also report that alisertib provided clinical benefit to this group of patients, over half of whom were heavily pretreated. These results are important given the limitations of current myelofibrosis therapies, including those of ruxolitinib, which include an average duration of response of 2–3 years. Second, alisertib restored normal morphology and GATA1 expression to the pathognomonic atypical megakaryocytes and further, reduced the degree of bone marrow fibrosis. The effects were generally associated with sustained clinical responses. A notable exception was patient 01-02 whose marrow showed a decline in GATA1 staining and no improvement in fibrosis with treatment. Despite a sustained symptom and anemia response, this patient developed AML after 19 cycles. These data underscore the critical contributions of megakaryocytes to the fibrotic process in myelofibrosis and highlight the unique activity of AURKA inhibition on this lineage. Third, the results also suggest that atypical megakaryocytes are required for the maintenance of fibrosis. Together, these data provide a strong rationale for the further study of AURKA inhibition as a therapeutic option in myelofibrosis and provide direct evidence that megakaryocytes with activated JAK/STAT signaling promote bone marrow fibrosis in humans.

J.M. Watts reports receiving commercial research grants from Takeda, speakers bureau honoraria from Jazz, and is a consultant/advisory board member for Jazz, Pfizer, and Celgene. J.K. Altman is a consultant/advisory board member for Daiichi Sankyo, AbbVie, Theradex, GlycoMimetics, Agios, Novartis, Astellas, Celgene, Immune Pharmaceuticals, and Syros. M.M. Patnaik is a consultant/advisory board member for StemLine Pharmaceuticals. R.K. Rampal reports receiving commercial research grants from StemLine Pharmaceuticals and Constellation, and is a consultant/advisory board member for StemLine Pharmaceuticals, Celgene, Jazz, Partner Therapeutics, Agios, and Blueprint. B. Stein is a consultant/advisory board member for Incyte and Apexx Oncology. J.D. Crispino reports receiving commercial research grants from Scholar Rock and Forma Therapeutics, and is a consultant/advisory board member for Sierra Oncology and MPN Research Foundation. No potential conflicts of interest were disclosed by the other authors.

Conception and design: R. Swords, Y.T. Dinh, F.J. Giles, B. Stein, J.D. Crispino

Development of methodology: R. Swords, Y.T. Dinh, F.J. Giles, B. Stein

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): N. Gangat, C. Marinaccio, J.M. Watts, S. Gurbuxani, J.K. Altman, Q.J. Wen, C.A. Famulare, A. Patel, R. Tapia, A. Handlogten, D. Zblewski, M.M. Patnaik, A. Al-kali, Y.T. Dinh, K. Englund Prahl, S. Patel, J.C. Nobrega, D. Tejera, A. Thomassen, J. Gao, R.K. Rampal, F.J. Giles, A. Tefferi, B. Stein

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): N. Gangat, C. Marinaccio, R. Swords, J.M. Watts, A. Rademaker, A.J. Fought, Q.J. Wen, N. Farnoud, R.R. Vallapureddy, A. Handlogten, M.M. Patnaik, A. Al-kali, Y.T. Dinh, D. Tejera, A. Thomassen, P. Ji, R.K. Rampal, F.J. Giles, B. Stein, J.D. Crispino

Writing, review, and/or revision of the manuscript: N. Gangat, C. Marinaccio, R. Swords, J.M. Watts, A. Rademaker, A.J. Fought, O. Frankfurt, J.K. Altman, N. Farnoud, M.M. Patnaik, A. Al-kali, Y.T. Dinh, D. Tejera, J. Gao, R.K. Rampal, F.J. Giles, A. Tefferi, B. Stein, J.D. Crispino

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): N. Farnoud, C.A. Famulare, A. Graf, A. Handlogten, Y.T. Dinh, K. Englund Prahl, D. Tejera, F.J. Giles

Study supervision: R. Swords, Y.T. Dinh, K. Englund Prahl, D. Tejera, B. Stein, J.D. Crispino

Other (clinical research coordinator for trial): S. Barath

This research was funded by a Translational Research Program grant from the Leukemia & Lymphoma Society (R6480-15) to J.D. Crispino (N. Gangat, C. Marinaccio, R. Swords, J.M. Watts, A. Tefferi, and B. Stein were supported by the grant). This work was also funded by the Samuel Waxman Cancer Research Foundation with support to J.D. Crispino. Alisertib was provided by Takeda Pharmaceuticals.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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