Final results of a phase 2 clinical trial of LCL161, an oral SMAC mimetic for patients with myelofibrosis

Myelofibrosis (MF), one of the myeloproliferative neoplasms (MPNs), is a clinically aggressive hematologic malignancy marked by anemia, thrombocytopenia, hepatosplenomegaly, and a predisposition to transformation to acute myeloid leukemia (AML) in higher risk patients.¹ Strikingly, survival outcomes (median overall survival [OS], 13-28 months] remain poor for patients with chronic-phase disease in whom frontline JAK inhibitor therapy has failed and subsequent clonal evolution will develop.^2-4 One of the most challenging aspects of treatment of patients with MF is that many patients have severe anemia and related consequences, as both an aspect of the underlying MF itself, and also, often related to administration of JAK inhibitors.^5-8 Furthermore, thrombocytopenia in MF (especially platelets below 50 × 10³/μL), which is regarded as an independent poor prognostic marker, also represents a therapeutic challenge, as most JAK inhibitors are difficult to administer or are contraindicated in this setting.⁹ Therefore, investigation of novel agents beyond JAK inhibitors is urgently needed to provide additional therapeutic options for patients with MF.^10,11 

Evasion of apoptosis is one of the key features of cancer cells.¹² One novel class of therapeutics that can restore apoptotic cell death in cancer cells is the second mitochondrial activator of caspases (SMAC) mimetics, also known as antagonists of inhibitors of apoptosis (IAP).¹³ Small-molecule drugs that can overcome the IAP-mediated apoptosis resistance of cancer cells have been identified.^14,15 

Therapeutic targeting of members of the IAP superfamily was investigated in patients with advanced solid tumors^16,17 and hematologic malignancies.^18-25 Infante et al were the first to investigate the oral, monovalent, SMAC mimetic LCL161, in a first-in-human, large phase 1, dose-escalation clinical trial in patients with advanced solid tumors, demonstrating overall safety and feasibility of administering this once-weekly, oral medication. Furthermore, upon correlative study in this clinical trial, LCL161 showed downmodulation of cellular IAP1 (cIAP1), demonstrating, as hypothesized, an on-target effect of this novel agent.¹⁷ 

LCL161, by binding to and degrading cIAP1 and, to a lesser extent, cIAP2 and XIAP, is involved in activation of noncanonical NF-κB signaling, and likely upregulation of inflammatory cytokines, resulting in both caspase- and tumor necrosis factor α (TNFα)–mediated apoptotic cell death.²⁶ Preclinical data demonstrate that overexpression of TNFα may act as a central mediator of clonal dominance of JAK2V617F-expressing MPN cells.²⁷ Furthermore, Heaton et al demonstrated that TNFα is overexpressed by MF CD34⁺ cells, that the TNFα/NF-κB pathway is upregulated in MF bone marrow cells, and that the increase is associated with elevated cIAPs and impaired selective inhibition of TNF receptor 2–mediated cell death.²⁸ We therefore hypothesized that LCL161 would disrupt this survival signaling by inducing cIAP1 degradation in the presence of TNFα and promote formation of a death-inducing signaling complex and apoptosis, thus leading to inhibition of clonal activity and clinical benefit in patients with MF.

We conducted an investigator-initiated, single-center, phase 2 clinical trial with oral, once-weekly LCL161 in patients with MF. The primary objective was to evaluate the efficacy and objective response rate (ORR) by International Working Group-Myeloproliferative Neoplasms Research and Treatment (IWG-MRT) 2013 criteria.²⁹ Secondary objectives were evaluation of safety, time to response, duration of response, and assessment of the burden of MPN symptoms (Myeloproliferative Neoplasm Symptom Assessment Form-Total Symptom Score). Correlative studies included evaluation of cIAP1/2 and XIAP levels and extended next-generation sequencing (NGS) of additional molecular mutations.

Patients aged ≥18 years, with Eastern Cooperative Oncology Group Performance Status (PS) of 0 to 2 and a confirmed diagnosis of post–essential thrombocythemia, post-polycythemia, or primary MF were enrolled. Patients had to have intermediate or high-risk MF by the International Prognostic Scoring System (IPSS) and be deemed not to be candidates for, intolerant to, or treatment-resistant to JAK inhibitor therapy. Importantly, there was no baseline minimum platelet count for study entry, and patients with prior allogeneic stem cell transplantation (SCT) were enrolled. A 2-week washout period from prior MF-directed therapy was necessary before the study treatment began. This clinical trial was approved by the MD Anderson Cancer Center Institutional Review Board, and each patient signed an informed consent, in accordance with the Declaration of Helsinki.

LCL161 was previously studied in a large, solid-tumor, phase 1 clinical trial.¹⁷ In that study, the authors investigated a variety of dose levels. In the 53 patients treated, oral doses ranged from 10 to 3000 mg weekly. The occurrence of 1 episode of cytokine release syndrome (CRS) was the dose-limiting toxicity, and although a formal maximum tolerated dose was not established, the 1800-mg oral dose was selected as a possible dose to move forward in further clinical trials. Out of an abundance of caution for the possibility of CRS and given that oral LCL161 has pharmacokinetic/pharmacodynamic data for activity at lower doses than 1800 mg weekly, we designed the MF study to start at 1 dose level lower: 1500 mg weekly.

We enrolled patients in the study from January 2015 through September 2019. We administered LCL161 orally once weekly, with a starting dose of 1500 mg, with 2 built-in dose level (DL) reductions (DL-1, 1200 mg; DL-2, 900 mg). Cycle length was 28 days. After the first 3 cycles, and every 6 cycles thereafter, a bone marrow biopsy and response evaluations were performed. In addition, we included an option to add very-low-dose steroids (2 mg oral dexamethasone) as a premedication before each weekly dose, as determined by the primary investigator.

Peripheral blood was collected from the first 40 patients at C1D1, C2D1, and C3D1, if the patients were available. Mononuclear cells were isolated from the samples by density gradient centrifugation with Lymphocyte Separation Medium (Corning, Manassas, VA). Protein levels were determined by western blot analysis. Antibodies for cIAP1 and cIAP2 were purchased from R&D Systems (Minneapolis, MN) and the antibody for XIAP from BD Biosciences (San Jose, CA). The Odyssey Infrared Imaging System was used for signal detection, and Odyssey software version 3.0 was used for signal quantification (LI-COR Biosciences, Lincoln, NE). Protein levels during the treatment (C2D1 and C3D1) are compared with those of the baseline levels (C1D1). Patient baseline protein levels are relative to those in OCI-AML3 cells, which served as positive controls and were included in all western blot protein measurements. β-Actin was used as a loading control.

All patients were registered through the Clinical Oncology Research System at MD Anderson Cancer Center. The trial was designed with a safety lead-in, in which 3 to 6 patients were designated to be treated at the 1500 mg dose first. If at least 2 patients of those treated first had experienced dose-limiting toxicity at a dosage of 1500 mg during the first cycle, the trial would have been halted for further investigation.

The primary efficacy end point was objective response (OR), defined as CR (complete remission) + PR (partial remission) + CI (clinical improvement). The primary objective of this study was to assess efficacy measured by the ORR after at least 3 cycles of treatment. A Simon 2-stage design was implemented. The target ORR was 35%, and a response rate of 18% or lower was not considered worthy of further investigation. With a type 1 error rate of 4% and 80% power, we planned to enroll 16 patients in the first stage. If ≤3 patients achieved OR, the trial would be stopped. If ≥4 of the first 16 patients responded, accrual would continue until 50 patients had been treated. After enrollment of the first 16 patients, the study would not enroll a new patient until enough responses (at least 4 responses in 16 patients) were observed to warrant continuation of the study.

At the end of the study, if ≥14 of 50 patients responded, the treatment would be considered efficacious and worthy of further investigation. Under this 2-stage design, the probability of early termination was 68% if the true ORR was 18%. NCSS-PASS 2005 software was used to create the statistical design details.

The method of Thall et al was used for toxicity monitoring³⁰ in the 50 patients treated. Specifically, the trial would be stopped for new patient enrollment if at any time during the study there was a more than 90% chance that the toxicity rate would be more than 30%. Summary statistics were used for continuous variables. Frequency tables were used to summarize categorical variables. The ORR was estimated, along with the Bayesian 95% credible interval. The Kaplan-Meier method was used to assess overall survival (OS), event-free survival (EFS), time to response, and duration of response. OS is defined as the time from the start of treatment until death of any cause or the time of last follow-up, whichever occurred first. EFS is defined as the time from the start of treatment until disease progression, death of any cause or the last follow-up. Data from all patients who received any study drug were included in the safety analyses. Toxicities were graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events, version4.03, whenever possible. We followed standard reporting guidelines for adverse events (AEs). Safety data were summarized by category, severity, and frequency. The proportion of patients with AEs was estimated, along with the Bayesian 95% credible interval.

A total of 53 patients were enrolled in the study. Fifty patients actually received at least 1 dose of study drug and were therefore eligible for and included in the safety and efficacy analyses (3 patients were withdrew before receiving any dose of the study drug: 1 screening failure caused by the need for urgent surgery; 1 withdrew consent; and 1 was concomitantly diagnosed with mantle cell lymphoma along with MF during the screening period). Thus, 50 patients constituted the study treatment group for overall analysis.

Median age was 72 (range, 56-85 years). Fifty-six percent were male. The median baseline platelet count was 52 ×10³/μL (range, 6-1365). The median baseline spleen size was 12 cm below the left costal margin by palpation in 30 patients with an enlarged spleen at baseline. Sixty-six percent of the patients had received ≥2 prior therapies, 56% had prior JAK inhibitor therapy, and 2 patients had prior SCT. Notably, 74% of the patients were IPSS-classified as high risk. Patient characteristics are in Table 1. Among the 3 most common MPN driver mutations, we observed JAK2 V167F (64%), CALR (12%), and MPL W515L (12%); 5 patients had triple-negative MF. Further mutational analysis by NGS revealed the 3 most common additional mutations: ASXL1 (28%), TET2 (15%), and DNMT3a (11%). See Table 2 for details of cytogenetic and molecular abnormalities.

All 50 patients treated were included in the safety analysis. The most common grade 1 and 2 nonhematologic AEs observed during the study were N/V (64%), fatigue (46%), and dizziness/vertigo (30%). The most common grade 3 and 4 nonhematologic AEs were syncope (n = 2; 4%), N/V (n = 1; 2%), and skin eruption/pruritis (n = 1, 2%). In terms of hematologic toxicity, the most common grade 3 or 4 events were thrombocytopenia in 3 patients (6%) and anemia in 2 (4%). Importantly, no CRS events were observed. There were 2 deaths during the course of the study, both deemed to be unrelated to the study drug (1 patient, an 84-year-old woman, who had been in the study for 35 cycles and had achieved CI of symptoms for 8.4 months, died of an intracranial hemorrhage in the setting of baseline thrombocytopenia. The second patient, a 78 year-old man, the final patient enrolled in study, died after 3 cycles in the setting of respiratory failure that was most likely caused by an underlying disease state and comorbidities and was unrelated to the study drug). Table 3 shows details of the AEs.

The protocol allowed for up to 2 levels of dose reductions during the course of the study: DL-1 (1200 mg oral administration weekly) and DL-2 (900 mg weekly). Overall, 18 of 50 (36%) patients required dose reductions. The most common cause of the reduction was a distinct grade 2 fatigue syndrome (n = 10) that appeared to be different from the baseline MPN symptom of fatigue. After dose reduction, 2 of 18 (11%) of those patients remain in the study, with 2 of 2 (100%) showing an ongoing IWG-MRT response. The dose was reduced in 5 patients on 2 occasions, down to the 900-mg oral weekly dosing schedule; 1 of 5 (20%) remained in the study at 900 mg per week.

All responses were evaluated in accordance with the revised IWG-MRT 2013 criteria, which calls for a response to last for ≥12 weeks.²⁹ The MPN symptoms were was evaluated serially in all patients using the Myeloproliferative Neoplasm Symptom Assessment Form-Total Symptom Score.³¹ All 50 patients treated with the study drug were included in this analysis; OR was 30% (15 of 50 treated patients) with 95% credible interval 0.18-0.43. A Swimmer plot for all responding patients is presented in Figure 1A. Fifteen patients exhibited 19 separate objective responses (4 patients met the criteria for 2 separate IWG-MRT 2013 responses). CI of symptoms (n = 11), CI anemia (n = 6), CI spleen (n = 1), and cytogenetic response (n = 1; Figure 1B). Determination of spleen size was performed serially by physical examination/palpation. Figure 1C is a waterfall plot showing the percentage of change in the spleen. Of 30 patients with baseline splenomegaly (spleen at least 1 cm), 22 patients had spleen reduction to some degree, whereas 1 patient had no change, and 7 had spleen enlargement. One patient had CI spleen by strict IWG-MRT 2013 criteria. At a median follow-up of 22.2 months (range, 7.9-60.1+), the median number of cycles received was 6 (1-64+), the median time to response was 1.4 months (0.9-9.1), and median response duration was 31.5 months (3.6-59.2+). The median cumulative dexamethasone premedication dose (given as 2 mg orally once weekly) per patient was 47 mg (range, 2-430 mg). There were 8 long-term (≥1 year) responders, with 6 of these long-term responders still in the study at ≥2 years. Importantly, for the overall cohort, the median OS was 34 months (range, 2.2-60.1+ months; Figure 2). In terms of molecular subtypes, we examined outcomes in patients with the JAK2V617F mutation vs JAK2V617F-negative patients. No statistically significant difference was observed in EFS or OS between the 2 groups. Among the 56% of patients who had received prior JAK inhibitors, the median OS was 33.4 months (range, 2.7-60.1). We next examined outcomes in patients harboring mutated ASXL1, as this high-risk mutation is commonly exhibited in MF and was detected in >25% of our patients. There were no statistically significant differences in either EFS or OS in patients with mutated ASXL1 vs those with wild-type ASXL1.

Of the patients with anemia, 6 achieved CI anemia (4, hemoglobin responses; 2, achievement of transfusion independence [TI]). Among those 6 patients, median time to response was 3.1 months (range, 0.9-9.1); median response duration was 8.4 months (range, 5.5-35; Figure 1D).

Overall, 43 of 50 (86%) of patients discontinued treatment. Reasons for withdrawal included stable disease/no objective response (n = 16); progressive disease (n = 11) [progression to AML (n = 5), progression of MF/splenomegaly (n = 6)]; patient choice/withdrawal of consent (n = 5); comorbidities (n = 4); proceeded to SCT (n = 2; 1 patient after 2 cycles, 1 patient after 6 cycles on therapy); toxicity (n = 2) (grade 2 fatigue in 1 patient; grade 3 syncope in 1); and 1 patient who experienced CI of symptoms with no other improvement and was withdrawn from the study. There were 2 deaths during the study period, both deemed to be unrelated to the study drug.

Protein levels of cIAP1, cIAP2, and XIAP, the targets of LCL161, were determined by western blot analysis in all available samples (up to patient 40) (Figure 3). Note that cIAP2 measurement was added later; hence, it was missing in samples from the patients enrolled early. Figure 3A shows the results of IAP levels in the long-term (>2 years) responders to the therapy (n = 7) (1 patient was missing C2D1 and 2 patients were missing C3D1 blood samples). Significant reductions in cIAP1 (mean ± standard error of the mean 0.50 ± 0.08, compared with 1.00 C1D1; P < .001) and cIAP2 (0.53 ± 0.08; P < .01) were achieved in all long-term responders at C2D1. Both cIAPs were either further reduced in some samples or remained at the decreased levels in others at C3D1 (0.19 ± 0.10 for cIAP1; P < .001, and 0.38 ± 0.07 for cIAP2; P < .01, compared with C1D1). For XIAP, the levels were decreased in some or largely unchanged in others (0.70 ± 0.14 for C2D1; P < .05 and 0.62 ± 0.23 for C3D1). In the short-term responders (responded then relapsed in <2 years; Figure 3B), similar to the long-term responders, marked decreases in cIAP1 levels were also observed at both C2D1 (0.43 ± 0.12; P < .01) and C3D1 (0.34 ± 0.02; P < .0001). Compared with the long-term responders, however, less cIAP2 reduction in some blood samples or no change in the others was found (0.92 ± 0.09 for C2D1 and 0.70 ± 0.10 for C3D1; P < .05). XIAP was either slightly decreased or increased (1.10 ± 0.28 for C2D1 and 1.07 ± 0.34 for C3D1). Interestingly, although cIAP1 was largely diminished in patients 8 and 37, XIAP was increased in both patients during the treatment (Figure 3B). In nonresponders (Figure 3C), although significant, less overall reduction in cIAP1 was observed (0.75 ± 0.10 for C2D1; P < .01, and 0.62 ± 0.10 for C3D1; P < .0001), but increases in cIAP2 (0.90 ± 0.003 for C2D1 and 0.96 ± 0.003 for C3D1) and XIAP (2.23 ± 1.03 for C2D1 and 3.52 ± 1.72 for C3D1) were found in many patients. Although several patients (such as 5, 9, and 22) achieved marked cIAP1 reduction, they also had great induction of XIAP during treatment, suggesting increased cIAP2/XIAP expression associated with the treatment resistance. These results suggest that sufficient reductions in cIAP1 and cIAP2 are likely to be necessary for patients to achieve and maintain responses. Insufficient reduction of cIAP2 and/or increases in XIAP relate to disease progression and resistance. The baseline (C1D1) IAP levels did not appear to correlate with responses (Figure 3D). However, the highest baseline levels of XIAP were observed in patients with resistance and disease progression (Figure 3D).

Serial analyses of available bone marrow tissue were performed. Overall, no major trend was observed in reduction of bone marrow fibrosis in responders.

This novel clinical trial represents the first large-scale analysis of patients treated with a SMAC mimetic, specifically in the field of MPNs. We demonstrated, in 50 patients with MF and a median age of 72 years, a median baseline platelet count of 50 × 10³/μL, 74% with a high-risk IPS, 28% with an ASXL1 mutation, 30% with an OR, and a median OS of 34 months.

One important aspect of the current study was the finding of 6 patients with anemia responses, which included 2 who achieved TI. The pursuit of anemia responses to novel agents remains an important aspect of drug development efforts in MF, and several agents with a focus on ameliorating the anemia of MF have shown early promise, including sotatercept³² and luspatercept.³³ In-depth correlative and mechanistic studies are warranted to further understand the mechanism by which LCL161 improved anemia in these 6 patients, especially in terms of modulation via inflammatory pathways including NF-κB and others.³⁴ 

Regarding biomarker development, measuring cIAP1 levels was shown to be feasible in the previous large solid tumor study.¹⁷ In the present study, on-target cIAP inhibition was also observed in all responders treated with LCL161 (Figure 3). Our data suggest that a sufficient decrease in cIAP1 is necessary for patients to achieve responses. Effective reduction in cIAP2 without induction of XIAP may be required for maintaining long-term responses. Although we did not find correlations of baseline IAP level with response, we observed that high baseline levels of XIAP and/or XIAP increase during therapy contributed to disease progression and resistance to LCL161 (Figure 3). This finding, if confirmed, could be hypothesis generating for further investigation of mechanisms of resistance to this novel class of agents and could serve to inform future combinatorial strategies.

The median OS in this study was 34 months, with response in several patients ongoing at ≥2 years in the study, and the longest enrolled patient at 5+ years ongoing response. Previous studies have shown median OS of ?13 to 14 months as a reasonable expectation for treatment-resistant patients with MF.^2–4 Furthermore, thrombocytopenia (platelet count, <100 × 10³/μL) is recognized as an independent poor prognostic risk factor in patients with MF⁹. The baseline median platelet count in this study was only 52 × 10³/μL.^9,35 We observed that 74% of the patients had a high-risk IPS at baseline, 56% had received prior JAK inhibitor therapy, 66% had received ≥2 prior therapies, and 2 had undergone prior SCT. It is of highest importance to evaluate and report on long-term overall survivals in each new non-JAK inhibitor clinical trial, as the field of treatment-resistant MF therapy moves forward, in addition to the traditional end points of spleen volume reduction and symptom burden benefit in this high-risk, treatment-resistant setting.

There were several limitations of this investigator-initiated study. As observed in the previous solid tumor study,¹⁷ a notable toxicity in our study was that of a distinct fatigue syndrome that resulted in 56% of all dose reductions in the study (10 of 18 patients). The fatigue was most commonly grade 2, and peculiarly, tended to last 24 to 48 hours maximum in most patients in whom it occurred. This, importantly, allowed for clinical differentiation from the baseline fatigue related to the underlying MF. Several patients who needed dose reductions, including those that were fatigue induced, have ongoing IWG-MRT responses. Further correlative studies are necessary to further assess this toxicity. It is critical to keep this side effect in mind and to consider the overlapping toxicities when combining LCL-161 with other classes of therapy. Another notable toxicity was that of dizziness, occurring in 30% of patients, making it the third most common nonhematologic toxicity encountered by the study patients and differing from the previous solid tumor study. Pertinent to this discussion and considering neurological activity and toxicity, there is preclinical evidence of activity of SMAC mimetics in glioblastoma model systems. Beug et al reported results of experiments with SMAC mimetics including LCL161, in which they observed activity in glioblastoma, and they drew the conclusion that, in a compromised blood-brain barrier state, SMAC mimetics could have activity and toxicity in neural niches.³⁶ 

In addition, because of this being an investigator-initiated study with limited funding, we were not able to include formal spleen computed tomography or magnetic resonance imaging for serial splenic measurements. As this is common in investigator-initiated studies in the field of MF, it did not affect the overall conduct, evaluation, or interpretation of the analysis results of this clinical trial. A final limitation of our study is the inclusion of both JAK inhibitor-resistant as well as JAK inhibitor-intolerant patients, as these terms have varying definitions across clinical trials; however, notably, there is precedent for these same criteria as the field enters into evaluation and response assessment in the novel, post-JAK inhibitor therapy era.^37–39 

Future directions for LCL161 and the SMAC mimetic class of therapies will include investigation for rational therapeutic combinations in patients with MPNs and related myeloid malignancies.⁴⁰ Several datasets have shown activity in different hematologic malignancies. There are conflicting data with regard to the prospects of combination with JAK inhibitors, with studies from Ramakrishnan et al showing possible synergism in multiple myeloma cell lines,²¹ whereas recent results from Craver et al showed that JAK inhibitors may not be the most optimal combination partner for LCL-161 in MPNs.⁴¹ As postulated by Craver et al in their preclinical work, a reason for the difficulty in combining the 2 classes of drugs may be that, because the kinase activity of JAK is needed for the sensitivity of JAK2 V617F–mutated cells, the addition of JAK inhibitors impairs the ability of SMAC mimetics to target the JAK2-mutated cells.⁴¹ Further studies in this area of SMAC mimetic plus JAK inhibitor are therefore needed to clarify whether this preclinical observation carries over into the clinic. Combination therapy with hypomethylating agents may be another avenue for research investigation, including investigating the combination of LCL161 with either azacytidine or decitabine. Borthakur et al initially studied such a combination with the bivalent intravenous SMAC mimetic birinapant in combination with azacytidine in patients with MDS.⁴² One of the most intriguing aspects of SMAC mimetics is their potential for immunomodulating effects, possible additive and/or synergistic activity with immunotherapy agents, and even as possible immune sensitizers in chimeric antigen receptor-modified T-cell approaches.^43–46 

In conclusion, we report the first study of patients with MF receiving weekly administration of a novel oral monotherapy, LCL161, a monovalent SMAC mimetic that showed both a safe profile and overall clinical efficacy in a large, high-risk, older study population.

This study was supported in part by National Institutes of Health (NIH), National Cancer Institute MD Anderson Cancer Center Support Grant (CCSG) grant CA100632 and NIH, Specialized Programs of Research Excellence (SPORE) grant CA016672 to the MD Anderson Leukemia Center. The authors thank Novartis for supplying the drugs and for support of this investigator-initiated clinical trial.

Contribution: N.P., B.Z.C., P.B., and S.V. designed and wrote the manuscript; W.Q. and X.W. supported the biostatistical trial design; N.P., B.Z.C., and P.Y.M. designed the correlative studies; and all authors contributed to enrollment of patients, data collection and analysis, laboratory/correlative analyses, and manuscript editing and evaluation.

Conflict-of-interest disclosure: N.P. has been a consultant to and received honoraria from Celgene, Stemline, Incyte Novartis, MustangBio, Roche Diagnostics, LFB, and Pacylex and has received research funding and clinical trial support from Stemline, Novartis, Abbvie, Samus, Cellectis, Plexxikon, Daiichi-Sankyo, Affymetrix, and the SagerStrong Foundation.

Correspondence: Naveen Pemmaraju, Department of Leukemia, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030; e-mail: npemmaraju@mdanderson.org.