Background
Therapeutic options for patients with acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), or advanced myeloproliferative neoplasms (MPNs) are still limited, and novel molecularly targeted therapies are needed. 5-Azacytidine (5-Aza) is a hypomethylating agent (HMA) [
1] commonly used as a lower intensity regimen in MDS, AML, and MPNs [
2,
3]. While 5-Aza has shown clinically meaningful responses and disease control [
2,
3], there remains a need to develop more effective and well-tolerated novel rational combinations [
4,
5]. Deletions or monosomies of chromosomes 5 and 7 are frequent in MDS and AML and portend a worse prognosis [
2,
3]. Genes on these chromosomal regions regulate tumor suppressor networks suggesting that these genes or chromosomal regions are involved in disease pathogenesis, which consequently would make them therapeutic targets either alone or with 5-Aza [
6]. Thus, silencing of relevant target genes would enhance 5-Aza activity and consequently make drugs targeting such genes candidates for combination with 5-Aza. Further, a haploinsufficient therapeutic context for patients with chromosome 5/7 aberrations can be discovered, if silencing of a specific gene within these commonly deleted regions (CDRs) can be effectively inhibited and sensitizes to 5-Aza. To identify such novel target genes and molecular vulnerabilities in AML, MDS, and MPNs, we performed an RNA-interference (RNAi) screen of 270 genes located within the CDRs of chromosomes 5 and 7, in combination with 5-Aza treatment. Several genes within the Hedgehog pathway (HhP) were identified as potential sensitizers to 5-Aza. Based on these and other pre-clinical observations of a potential role of the HhP in myeloid disease as highlighted below, a clinical phase 1/1b trial of 5-Aza with the smoothened (SMO) inhibitor LDE225 (erismodegib) in AML, MDS, and MPN patients was initiated and is currently accruing patients.
The HhP is highly evolutionarily conserved and plays critical roles in embryonic development, such as regulating patterning and limb formation, and is involved in the homeostasis of many human organs and tissues including in the hematopoietic system [
7]. HhP genetic alterations in the HhP are linked to the development of several human tumors such as basal cell carcinoma (BCC), medulloblastoma (MB), and rhabdomyosarcoma. Aberrant HhP signaling without evidence of genetic defects has also been linked to disease pathogenesis of many other tumors. The HhP can be activated by extracellular ligands, such as Sonic or Indian Hedgehog (SHH, IHH), that bind the transmembrane receptor Patched (PTCH) [
7]. PTCH, when active, constitutively inhibits SMO and thus downstream transcription factors like GLI-1, GLI-2, GLI-3, as well as other genes involved in cell proliferation or survival (e.g., BCL-2, BCL-X
L) [
7]. Activated HhP signaling, for example, by inhibitory mutations in PTCH in BCC (i.e., the PTCH “brake” is removed) [
8] or by overexpression or activation of SMO, contributes to malignant transformation via the aforementioned transcription factors and anti-apoptotic genes. Currently developed drugs, including erismodegib, mostly target/inhibit SMO as an essential intermediate gene within the HhP that is activated and mediates intracellular signaling.
A rationale for inhibiting HhP signaling in myeloid malignancies has been described in the literature based on observations that HhP signaling regulates erythroid progenitor cell proliferation and differentiation [
9] and is thought to be essential for the maintenance of myeloid cancer stem cells [
10]. For example, SHH activates downstream transcription factor GLI-1 in several hematological malignancies, with prevalent expression observed in AML and acute promyelocytic leukemia (APL) patients [
11]. HhP genes
SHH,
SMO, and
GLI-1 are upregulated in chronic myeloid leukemia (CML) patients and are further elevated in blast crisis as compared to chronic-phase CML [
12]. It is further hypothesized that developmental pathways such as the HhP play a role in the expansion of BCR-ABL-positive leukemic stem cells (LSCs) and may be responsible for residual disease after BCR-ABL targeted therapies [
12]. Similarly, there seems to be a role for the HhP in LSCs of acute leukemias [
10] and other neoplastic myeloid diseases such as MPNs [
13], including polycythemia vera (PV), essential thrombocytopenia (ET), and primary myelofibrosis (PMF). Although the HhP plays a role in normal hematopoiesis and morphogenesis, the pathway is mostly silenced in normal adult tissue but re-activated in an oncogenic state. This rather selective tumor tissue expression best explains the good tolerance of SMO inhibitors in the clinic, with many patients being treated for several years [
14,
15].
Preliminary clinical activity of single-agent SMO inhibitors in AML, MDS, and MPNs, including myelofibrosis (MF), has been demonstrated [
16]. Herein, we report pre-clinical data of the novel combination of 5-Aza and the SMO inhibitor LDE225 in myeloid malignancies as a possible novel combination in advanced myeloid malignancies.
Conclusions
RNAi screening of AML and MDS relevant genes located on chromosomes 5 and 7 in combination with 5-Aza yielded potential targetable molecular vulnerabilities. Several of the 5-Aza sensitizing gene hits are situated within the HhP. Experiments with SMO inhibitors in vitro and ex vivo pharmacologically validate the idea that the HhP could serve as a therapeutic target with HMAs in myeloid malignancies. This is of immediate translational relevance as several well-tolerated SMO inhibitors are clinically developed. Trials of SMO inhibitors and HMAs, either 5-Aza or decitabine (DAC), are currently ongoing in patients with AML, MDS, and MPNs. To our knowledge, this is the first report showing synergy between an SMO inhibitor and a HMA in primary AML and MDS samples, as well as in AML cell lines.
The HhP is highly complex and difficult to examine in vitro and even in vivo. Thus, detailed molecular analyses of potential underlying mechanisms for the sensitization effects were not performed in this study. Because clinical development is proceeding so rapidly, future biology will be best explored using actual samples from patients treated on study. To that end, we are prospectively collecting sequential samples from patients on an ongoing trial combining 5-Aza with erismodegib (NCT02129101).
Foregoing an investigation of the complex HhP biology pre-clinically, we will focus efforts on exploring the biology of HhP and SMO inhibitors in situ with actual samples from patients on trial. We will perform global RNA sequencing and complementary genomic assays on serially collected samples to assess baseline and transcriptional changes associated with clinical response to SMO inhibition in AML and MDS. We prefer this in situ approach, as the ultimate proof of effectiveness for a novel combination can only be shown in the clinic, and novel combinations are urgently needed for advanced MDS and elderly AML patients. Consequently, the goal of the present study was to provide preliminary evidence of the sensitization and interaction between 5-Aza and SMO inhibition in primary malignant myeloid cells ex vivo, which has not been reported previously. Furthermore, RNAi screens have inherent biases for both false positives and negatives (i.e., hits derived from off-target activity and genes missed by insufficient silencing); thus, there are possibly additional target genes on the CDRs of chromosomes 5 and 7 that may sensitize to HMAs. Despite these limitations, RNAi screens can serve as a valuable assay to identify leads that can guide further pre-clinical validation and translation into the clinic.
Several observations regarding the sensitization between 5-Aza and SMO inhibition are worth noting. First, sensitization is at least partially independent of surrounding stroma cells and cellular structures indicating that HhP inhibition with SMO inhibitors in myeloid cells is at least partially cell autonomous (i.e., autocrine) or paracrine between malignant myeloid cells. Second, not all specimens showed sensitization in ex vivo assays. Sensitization was observed in approximately 30 to 50 % of samples making biomarker studies with actual patient samples even more important once outcome and clinical response assessments are available. Within the clinical characteristics of the patient samples examined ex vivo in this study, there was no apparent feature differentiating responding (sensitized) samples, based on cytogenetics, disease subtype, or targeted mutation profiling, from non-responding patient samples (Table
3). HhP gene activation appears to associate with single-agent sensitivity to SMO inhibitors (Additional file
3: Figure S1), whereas there was no apparent association with combination synergy. Limited pre-clinical in vitro data showed that concurrent treatment of erismodegib together with 5-Aza may be effective, which has informed trial design by adding a treatment arm with concurrent dosing of 5-Aza and SMO inhibitors. Concurrent treatment may allow SMO inhibitors to be further escalated to a dose that may otherwise not be tolerated if given continuously. We are exploring this concept in the ongoing trial (R. Tibes, personal communication).
In conclusion, targeting the HhP by inhibiting SMO, in combination with the HMA 5-Aza, shows sensitization in some, but not all, primary AML, MDS, and MPN patient samples. The mechanism(s) of synergy remain uncertain and require further investigation in future studies. Given the overall good clinical tolerance of SMO inhibitors, the activity of 5-Aza in MDS and AML, and pre-clinical studies presented herein, the rational combination of erismodegib and 5-Aza is being examined in an ongoing clinical trial.
Authors’ contributions
RT and JMB contributed to the experimental design, interpreted the data, and wrote the manuscript. GRO analyzed the data and helped in writing the manuscript. DD and NH performed the experiments, analyzed the data, and contributed to the manuscript writing. KB and JM helped with the experimental design, in performing the experiments, and in analyzing the data. AA, RAM, and JF helped in the study design, provided essential resources, and contributed to the manuscript writing. FR performed the cDNA library construction for NGS experiments. TW performed the DNA sequencing on the Illumina 2500. All authors read and approved the final manuscript.
RT is a physician-scientist developing novel therapies and researching rational combinations for patients with MDS and AML. RAM is a clinician investigator focusing on myeloproliferative neoplasms, new drug development, and patient-reported outcomes. JMB is a translational research scientist pursuing pre-clinical and clinical correlative studies to develop therapies and predictive biomarkers for the treatment of myeloid malignancies. TW is a Technical Specialist in the Mayo NGS core laboratory performing Next Gen Sequencing on all Illumina platforms. AA is a clinical researcher whose main focus is to bring newer and novel therapies to patients with MDS and AML. GRO is a bioinformatics lead working primarily on individualized medicine initiatives within the areas of cancer and inherited disease. KB has a Masters in Clinical Research Management and works on biomarker projects.