The Hedgehog pathway as targetable vulnerability with 5-azacytidine in myelodysplastic syndrome and acute myeloid leukemia

RNAi screens of genes from commonly deleted regions of chromosomes 5/7 with 5-azacytidine

To identify rational combinations with 5-Aza, a custom collection of small interfering
RNA (siRNA) targeting 270 genes selected from the CDRs of chromosomes 5 and 7 was
evaluated together with 5-Aza in RNAi sensitizer screens. Using four human myeloid
leukemia cell lines (TF-1, HEL, THP-1, and MDS-L), several HhP genes were found to
enhance 5-Aza activity. Specifically, three HhP genes emerge as sensitizers to 5-Aza
when silenced by siRNA in three of four cell lines examined (Fig. 1). SHH silencing sensitized to 5-Aza in both TF-1 and THP-1 cells, while SMO and GLI-3
silencing sensitized in HEL and THP-1 cells, respectively. The RNAi screen performance
was robust with high transfection efficacy (TF-1, 74–95 %; HEL, 58–86 %; THP-1, 89–96 %;
MDS-L, 49–91 %), and except for THP-1, little non-specific toxicity (i.e., the non-targeting
siRNA toxicity; for TF-1, 0–11 %; HEL, 0–20 %; THP-1, 34–70 %; MDS-L, 3–18 %). These
performance characteristics are comparable with other RNAi screens in myeloid cells
as we have reported previously 17], 18]. The effective concentration (EC) values (i.e., the percent reduction in relative
cell number) for 5-Aza ranged between ~EC
₅
and EC
₅₅
(Fig. 1 and Additional file 1: Table S1 A–D).

Fig. 1. RNAi screening hits in four AML cell lines (TF-1, HEL, THP-1, MDS-L). Venn diagrams
show the gene hits per screen and cell line. Hit definition/selection is described
in the “Methods” section. ****3 of 3 siRNA 2 stdev; ***2 of 3 siRNA 2 stdev; **1
siRNA 2 stdev and 1 siRNA 1 stdev; *2 or 3 siRNA 1 stdev. EC effective concentration of 5-azacytidine at which screen was performed

SMO inhibitors combined with 5-azacytidine in AML cells

Next, we examined the effect of two clinically developed SMO inhibitors, GDC-0449
(vismodegib), the first and only in-class approved SMO inhibitor 19], and LDE225, which is in clinical development and currently used in a combination
trial with 5-Aza (clincialtrials.gov: NCT02129101). EC
₅₀
values for LDE225 ranged from 3.8 to 15.2 ?M, and EC
₅₀
values for vismodegib ranged from 12 to 83 ?M (Table 1). Synergy between 5-Aza and LDE225, as calculated by the Chou-Talalay method, was
observed in seven molecularly heterogeneous AML cell lines with combination index
(CI) values from 0.48 to 0.71 (Table 2). Importantly, synergy is observed at 5-Aza concentrations of 0.8–2.5 ?M (Additional
file 2: Table S2), comparable to clinically achievable 5-Aza concentrations in humans. LDE225
concentrations corresponding to optimal synergy were mostly in the range of 4–8 ?M,
although both higher and lower LDE225 dose outliers are observed, as low as 0.25–0.5 ?M.

Table 1. Single-agent activity of SMO inhibitor in AML. Micromolar (?M) EC
₅₀
values of SMO inhibitors LDE225 (erismodegib) and GDC0449 (vismodegib) in AML cell
lines

Table 2. Synergy between 5-azacytidine and erismodegib in AML. Sensitization and synergy between
LDE225 (erismodegib) and 5-azacytidine (5-Aza). EC
₅₀
values in micromolar (?M) and synergy presented as combination index (CI) values

To determine if transcript expression of relevant HhP genes correlated with single-agent
activity or synergy, we assessed HhP signaling genes (e.g., SMO, SHH), HhP transcription factors (GLI-1, GLI-2, and GLI-3), and representative transcriptional
target genes (e.g., anti-apoptotic BCL-2 and BCL-XL [BCL2L1] and CDK1) for their expression in the four AML cell lines used in RNAi screening experiments
(TF-1, HEL, MDS-L, and THP-1), and enriched the dataset with an additional cell line
(MV4-11) for which RNA sequencing (RNAseq) data and drug treatment data was available.
Overall, there was no specific distinguishing expression signature; however, general
HhP activation (i.e., HEL and MDS-L) was associated with reduced sensitivity to single-agent
SMO inhibition. The synergistic effects (CI values), however, were independent of
HhP gene activation (Additional file 3: Figure S1), possibly suggesting that inhibiting SMO expression is more relevant
in combination with 5-Aza. These gene expression experiments also indicate that different
myeloid cells activate the HhP via modulation of distinct components of the HhP. For
example, MDS-L displayed highest expression of IHH, PTCHD 1/2/4, and GLI1/2, whereas
HEL preferentially expressed SMO and GLI2, THP-1 SHH, and SMO and GLI3. Expression
levels of genes are also consistent with RNAi hits in the respective cell lines (i.e.,
SMO in HEL, GLI3 and SHH in THP-1). Potential differences in HhP signaling will be
examined in samples from patients on trial using RNAseq as part of the biomarker analysis
(clincialtrials.gov: NCT02129101).

Hedgehog pathway inhibition in combination with 5-azacytidine in clonogenic assays

Strong sensitization with greatly reduced colony count is observed with combined LDE225
and 5-Aza as compared to either single agent in several primary MDS and AML samples
in clonogenic assays (Fig. 2, i.e., AMML#2, MDS#7, PV+MPN#1). However, some samples showed a neutral response
to the combination compared to one or both single agent (i.e., AML#1, AML#2, MDS#3),
while a few samples do not show added benefit over the combination, and may even exhibit
slight antagonism with combination treatment compared to either single agent (i.e.,
MDS#4, MDS#5). The greatest degree of sensitization is seen at 2–4 ?M of LDE225, at
a 5-Aza dose kept constant at 1 and 2 ?M to reflect clinically achievable concentrations
of 5-Aza. Interestingly, one sample (MDS#3) assessed at the time of MDS diagnosis
showed little benefit from the combination, yet a sample from this same patient drawn
and assayed at the time of progression to AML (AML#2) showed increased benefit from
both single-agent LDE225 and the LDE225/5-Aza combination. The clinical data and molecular
characteristics of patient samples examined in ex vivo clonogenic assays are shown
in Table 3. There was no correlation in this dataset to any obvious clinical molecular-cytogenetic
characteristics or to targeted sequencing of mutations in myeloid-associated genes
performed on a number of samples.

Fig. 2. Clonogenic growth inhibition of primary MDS and AML specimens. LDE225 (erismodegib)
and 5-azacytidine in primary patient samples. UT untreated. Dose of 5-Aza and LDE225 given in micromolar at y-axis. Percent growth at x-axis

Table 3. Clinical and molecular patient characteristics

Sequencing schedules of combined 5-azacytidine and SMO inhibitors and specificity
to HMA

As the optimal sequence of SMO inhibition with 5-Aza is not determined, drug sequencing
experiments using high, middle, and low LDE225 concentrations were performed on standard
doses of 5-Aza in vitro. As shown in Table 4, concurrent treatment at higher doses of 8 and 32 ?M LDE225 showed greatest sensitization
by fold-shift in EC
₅₀
values of 5-Aza, while sequential dosing showed a trend towards antagonism, at least
in vitro.

Table 4. Sequential versus concurrent 5-azacytidine/LDE225 treatment

A comparison of the synergistic potential of erismodegib with 5-Aza versus cytarabine
(Ara-C), the most commonly used AML cytotoxic drug, shows that while sensitization
is observed with 5-Aza, antagonism is observed when combined with Ara-C, demonstrated
by a rightward curve shift to a higher EC
₅₀
value (Additional file 4: Figure S2).