Role of activated MAPK pathway in mediating resistance to PI3K inhibitors in patients with chronic lymphocytic leukemia

Treatment for patients with chronic lymphocytic leukemia (CLL) has been transformed by inhibitors of Bruton’s tyrosine kinase and phosphatidylinositol 3-kinase (PI3K) that target the B-cell receptor signaling pathway. The genetic inactivation of the p110ẟ isoform of the PI3K catalytic subunit has been documented to cause impaired B-cell receptor signaling and B-cell migration, delaying the onset of CLL. Although several PI3Kẟ inhibitors have been approved, there are limited data on the mechanisms of acquired resistance to PI3Kẟ inhibitors.

The mitogen‑activated protein kinase (MAPK) pathway is a key signaling pathway that regulates a wide variety of cellular processes. Though MAPK activation is common in CLL, there is marked heterogeneity in the degree of its activation, and little is known about how these differences in MAPK activation affect responses to targeted therapy. Murali et al.¹ recently published a study in Blood investigating how resistance to PI3K inhibitors can be mediated by activation of the MAPK pathway. The key findings are summarized here.

Methods

Whole exome sequencing was performed on matched tumor and germline samples from 28 patients with CLL who had been treated with PI3K inhibitors using OSU-CLL and MEC1 cell lines.

High-confidence somatic mutations were identified using a set of algorithms within the Firehose pipeline developed by the Cancer Genome Analysis group.

Baseline characteristics

The median age of patients was 54 years (age range, 31–69 years) and 82% of patients were male. Patients were heavily pretreated with a median of 4.5 prior therapies. A median of 2 longitudinal tumor samples (range, 1–6) were sequenced per patient, with a total of 68 samples. Table 1 shows the baseline characteristics of all enrolled patients.

Table 1. Baseline characteristics*

Results

Genomic landscape at first sampling

A total of 28 patients with CLL were included in the study and divided into 18 responders (47 samples) and 10 non-responders (21 samples) based on their initial clinical response to the PI3K inhibitors. With an average of 23 ± 12 non-silent mutations, the frequency of non-silent mutations was 0.64 ± 0.32 per Mb (range, 0.11–1.61).

There was no difference in the median number of silent (p = 0.41) and non-silent (p = 0.72) mutations between responders and non-responders. Similarly, no differences were seen in the median number of silent (p = 0.64) and non-silent (p = 0.56) mutations between initial time point and the samples with the lowest mutations at the later time points. Mutation frequencies were higher than previously reported in the significantly mutated genes: BRAF, 11% vs 4% (p = 0.0999); DDX3X, 14% vs 2% (p = 0.0022); SF3B1, 36% vs 21% (p = 0.0641); and TP53, 21% vs 7% (p = 0.0116).

Distribution of driver and PI3K pathway mutations

BRAF (n = 5), KRAS (n = 2), MAP2K1 (n = 2), XPO1 (n = 1), PLEKHA1 (n = 1), NXF1 (n = 1), and INPPL1 (n = 1) were exclusively mutated in non-responders. MAPK pathway mutations (KRAS, BRAF, and MAP2K1) were identified in 60% of non-responders and 6% of responders who carried a NRAS mutation (p < 0.005). Among the five patients with a BRAF mutation, three had the mutation at the time of first sampling, one acquired it with therapy, and one showed a marked increase in cancer cell fraction of the BRAF clone with therapy.

Cancer cell fraction values for MAP2K1 (p = 0.01) and PLEKHA1 (p < 0.001) mutations increased with time, potentially indicating positive selection. Nine of the ten significantly enriched gene sets that overlapped with dynamic subclones were associated with cancer or ERBB2 signaling that contained MAPK pathway genes. Enriched genes were observed with extracellular-signal-regulated kinase (ERK) activation at the post-progression time points (p < 0.001) in both responders (n = 3) and non-responders (n = 2).

AKT and ERK activation status in PI3K responders and non-responders

Idelalisib inhibited AKT phosphorylation (pAKT) in both responders and non-responders (p < 0.001 in each). On the contrary, ERK phosphorylation (pERK) by idelalisib was significantly reduced in responders (p < 0.01) compared with non-responders (p = 1.00). Metabolic activity and ERK signaling at both responding and progressing time points was decreased with MAPK/ERK kinase (MEK)1/2 inhibitors. ERK1/2 inhibitor combined with idelalisib showed significant reduction in metabolic activity.

Activating mutations in MAP2K1 confer resistance to PI3K inhibitors

Mutated MEC1 (MEC1^mut) cells with E203K, F53L, and Q56P mutations in MAP2K1 showed increased pERK compared with MEC1 wild type (MEC1^wt) cells in both unstimulated and anti-immunoglobulin M‑stimulated conditions (all p < 0.001). pERK levels in MEC1^wt and MAP2K1E203K cells were inhibited by idelalisib (p < 0.01); however, MAP2K1F53L and MAP2K1Q56P cells were resistant to pERK inhibition by idelalisib. In OSU-CLL cells, only WT cells were affected by inhibition of pERK but not mutated OSU‑CLL (OSU^mut) cells.

On the contrary, pAKT was significantly inhibited by idelalisib in both MEC1^mut and MEC1^wt cells under anti-immunoglobulin M-stimulated conditions (p < 0.001 for both), as well as in OSU^mut and OSU^wt cells.

Inhibition of MAPK pathway signaling restores sensitivity to idelalisib in MEC1 cells

MEC1^wt and MEC1^mut cells showed a substantial reduction in pERK levels when treated with MEK1/2 inhibitor or ERK1/2 inhibitor (p < 0.001 in MEC1^wt cells and MEC1 cells with E203K, F53L, and Q56P MAK2K1 mutations). However, the mutant cells did show a substantial reduction in pERK when treated with idelalisib alone (E203K, p = 0.33; F53L, p = 0.67; Q56P, p = 0.61).

Idelalisib either alone or in combination with MEK1/2 or ERK1/2 inhibitor substantially reduced pAKT levels in both MEC1^wt and MEC1^mut cells (p < 0.001 in MEC1^wt, E203K, F53L and Q56P cells).

MEC1^mut and OSU^mut cells have attenuated sensitivity to idelalisib which is reversed by MAPK pathway inhibition

MEC1^mut cells showed significantly higher cell counts at 72 hours compared with MEC1^wt cells (E203K, p < 0.001; F53L, p < 0.01; Q56P, p < 0.001). Treatment of OSU-CLL and MEC1 cells with PI3K or MEK1/2 inhibitors significantly decreased the survival in both WT and mutant cells compared with their controls. However, when both WT and mutant cell lines were treated with idelalisib, the mutant cells were relatively resistant with both 5 µM (OSU^mut: E203K, p < 0.06; F53L, p < 0.001; Q56P, p < 0.001; and MEC1^mut: F53L, p < 0.01; Q56P, p < 0.01) and 10 µM idelalisib (OSU^mut: E203K, p < 0.01; F53L, p < 0.001; Q56P, p < 0.001; and MEC1^mut: F53L, p < 0.001; Q56P, p < 0.01).

Additionally, in the OSU-CLL cell line, MEK inhibitors directed a potent reduction in cell viability (WT, p < 0.001; E203K, p < 0.001; F53L, p < 0.001; Q56P, p < 0.001) but the combination of MEK1/2 and PI3K inhibition still had an additive effect in most cell lines. Single-agent MEK1/2 or PI3K inhibition in MEC1 cell lines showed diminished cell viability, but not significantly so. Greater reductions in cell viability were seen with the use of combined inhibitors (WT, p < 0.001; E203K, p < 0.001; F53L, p < 0.001; Q56P, p < 0.001).

Idelalisib (10 µM) led to a modest but significant decrease in proliferation in MEC1^wt cells (p < 0.05) compared with MEC1^mut cells (E203K, p < 0.05; F53L, p = 0.14; Q56, p = 0.43). Proliferation was only inhibited by idelalisib in OSU^wt cells (5 µM, p = 0.06; 10 µM, p < 0.05).

Conclusion

This study demonstrated that MAPK pathway mutations are drivers for primary resistance to PI3K inhibitors in patients with CLL. Furthermore, the study showed that persistent ERK1/2 activation mediates PI3K inhibitor resistance in CLL and treatment with MEK1/2 or ERK1/2 inhibitors, alone or in combination with idelalisib, diminishes the protective effects triggered by ERK1/2 activation. Taken together, the findings suggest that a combination of PI3K and MAPK pathway inhibitors at the start of therapy or as a supplement during early progression could help reduce PI3K resistance.