“All Our Wisdom is Stored in the Trees” - Degrading BCR-ABL with Berberis Vulgaris

In this issue of Clinical Cancer Research, Yin and colleagues demonstrate that berberine, a benzylisoquinoline alkaloid found in a variety of plants, induces degradation of both BCR-ABL and imatinib-resistant BCR-ABL–mutant T315I by recruiting E3 ubiquitin-protein ligase LRSAM1 (1).

First identified in by Peter Nowell in 1960, BCR-ABL is a chimeric oncogene generated by the translocation of sequences from the Abelson murine leukemia viral oncogene homolog 1 (ABL1) tyrosine kinase gene into the breakpoint cluster region (BCR). Alternative isoforms, p210BCR-ABL and p190BCR-ABL, are characteristic of chronic myeloid leukemia (CML; found in 95% of patients) and acute lymphocytic leukemia (ALL; found in 25%–30% of patients), respectively. The head to tail fusion of BCR to ABL1 causes loss of the negative regulatory SH3 domain on the tail of the ABL protein, which promotes autophosphorylation and constitutive ABL kinase activity. BCR-ABL activates various signaling pathways responsible for proliferation and survival, including Ras/MAPK, PI3K, and JAK-STAT pathways (2).

The discovery of tyrosine kinase inhibitor (TKI) imatinib to treat BCR-ABL–positive CML revolutionized not just CML treatment but cancer therapeutics as a whole. Upon FDA approval in 2001, TIME magazine hailed imatinib as the “magic bullet” in the fight against cancer. Five-year survival rates for patients with CML, which had previously hovered around 30% with standard chemotherapy, soared to 90% with imatinib treatment. Its success prompted a comprehensive shift in cancer therapeutics from cytotoxic therapies to molecularly targeted therapies, spurring the development of additional small-molecule TKIs targeting a wide array of oncogenic tyrosine kinases. Soon, however, patients who had achieved clinical remission on imatinib began to relapse. Sequencing of the BCR–ABL1 fusion gene from these patients demonstrated that many had developed point mutations in the ATP-binding pocket of ABL kinase that prevented imatinib from binding. The most prevalent among these was a single-nucleotide substitution in the ABL1 gene, resulting in a threonine to isoleucine substitution at amino acid 315 (T315I). Modeling of wild-type ABL kinase with imatinib predicted that although the substitution of isoleucine would still permit ATP binding, it would prevent imatinib binding by causing steric hindrance, as well as by eliminating a critical oxygen molecule needed for hydrogen bonding between imatinib and ABL (3).

Hoping to overcome the hurdle of acquired imatinib resistance, many sought to develop second-generation BCR-ABL kinase inhibitors. Among these, nilotinib and dasatinib showed the most promise. However, as with imatinib, the T315I mutation demonstrated complete clinical resistance to both compounds, with additional drug-resistant mutations arising in response to treatment. As such, third-generation inhibitor ponatinib was designed specifically to target the T315I point mutation. Thus far, ponatinib is the only approved BCR-ABL inhibitor that shows activity against T315I, although additional ponatinib-resistant mutations have arisen in response to treatment (4).

With acquired resistance a seemingly inevitable consequence to TKI therapy, Yin and colleagues focused their attention on the natural compound berberine as a novel targeted therapy for BCR-ABL–positive CML. Berberine is a benzylisoquinoline alkaloid found in plants belonging to Berberis, a large genus of deciduous and evergreen shrubs, that has a multitude of purported therapeutic applications. The antimicrobial activity of berberine has been well-established for some time and is used in Chinese traditional medicine to treat all manners of bacterial infections. The hypoglycemic effect of berberine was first observed when berberine was used to treat bacterial diarrhea in diabetic patients. Since then, berberine has been described as a treatment for type 2 diabetes, its mechanism being partially attributed to AMP-activated protein kinase activation and inhibition of protein tyrosine phosphatase 1B (PTP 1B). As an antineoplastic agent, berberine has shown activity against many high-risk cancers, including prostate, lung, and breast. Its antitumoral mechanism, however, remains largely elusive (5).

Berbamine, another Berberis-derived plant alkaloid, was previously shown to induce cell death in BCR-ABL–positive CML through binding to and inhibiting the kinase activity of Ca2+/calmodulin-dependent protein kinase II γ (CaMKIIγ; ref. 6). Given its chemical relation to berbamine, Yin and colleagues sought to identify putative molecular targets of berberine in BCR-ABL–positive CML cell lines using use surface plasmon resonance followed by high-performance liquid chromatography mass spectrometry. The authors identified a total of 28 proteins as putative berberine targets, including ABL kinase. Molecular docking studies demonstrate that berberine likely binds directly to the ABL kinase domain, suggesting that berberine may behave as a TKI. The authors validate that this binding is indeed a direct interaction using nuclear magnetic resonance followed by thermo shift assays, with just the kinase domain of ABL protein as input. They further demonstrate that berberine does indeed inhibit ABL kinase activity.

Importantly, the authors then go on to show that berberine has activity against not just BCR-ABL-, but BCR-ABL T315I-expressing cell viability in cell lines, mice, and primary human patient samples. To this point, the authors have essentially demonstrated that berberine can achieve what ponatinib can, by selectively killing first- and second-generation TKI-resistant BCR-ABL–mutant cells. As previously mentioned, however, ponatinib-resistant mutations develop in response to treatment, so to improve upon third-generation TKIs, berberine must overcome this impediment.

Upon the observation that expression levels of BCR-ABL protein were reduced in response to berberine treatment, the authors began to investigate the mechanism by which berberine induces BCR-ABL protein degradation. Indeed, this is a more critical and constructive finding than berberine as an ABL kinase inhibitor due to the prevalence of acquired ABL kinase inhibitor resistance. Remarkably, they found that berberine induces autophagy in BCR-ABL–positive cells, and that degradation of BCR-ABL occurs via the autophagic lysosome-related protein LRSAM1, an E3 ubiquitin ligase whose expression is upregulated by berberine (Fig. 1). This degradation is reversed upon treatment with autophagy inhibitors, suggesting that the recruitment of LRSAM1 and subsequent degradation is entirely autophagy dependent.

The importance of this finding cannot be understated. Complete degradation of the BCR-ABL oncoprotein has immense promise as a mechanism by which to overcome the acquired drug resistance inherent to TKI treatment. While the safety profile of berberine treatment for leukemia must be further evaluated, it represents a promising alternative to TKI therapy for BCR-ABL–positive CML and potentially ALL as well.

No potential conflicts of interest were disclosed.

S.E. Elf is supported by the National Heart, Lung, and Blood Institute (K99HL13692401A1), the Leukemia and Lymphoma Society Career Development Program Special Fellow Award, and the American Society of Hematology Scholar Award.