Cure rates for patients with AML have remained poor despite intensive chemotherapy and stem cell transplantation. For older adults, long-term survival is dismal with many older patients unable to tolerate standard cytotoxic therapy. Although much has been learned about the pathogenesis of AML, many of the potential targets involve abnormalities of transcription factors, a class of proteins considered “undruggable” with standard pharmacological methods. Emerging approaches, such as RNAi-based (Vorhies and Nemunaitis, 2009)
, gene expression-based (Lamb et al., 2006
; Stegmaier et al., 2007)
, stapled peptide technology (Walensky et al., 2004)
, or other peptide-based methods (Polo et al., 2004)
may ultimately enable targeting of this important protein class. Alternatively, the identification of more pharmacologically tractable targets in AML offers a parallel route to therapies for this disease.
In this work, we build upon the striking observation that multiple EGFR inhibitors had anti-AML activity by a non-EGFR mechanism and hypothesized that this was via a shared off-target effect. We integrate cross-disciplinary proteomic and genetic approaches to meet the difficult challenge of understanding the molecular basis for the biological activity of these EGFR inhibitors in AML. At the intersection of these two approaches, Syk was identified as a tyrosine kinase target in AML. Syk is a cytoplasmic tyrosine kinase widely expressed in hematopoietic cells and critical in B cell differentiation and signal transduction pathways. Syk is a member of the Syk/ZAP-70 family of nonreceptor kinases and is characterized by two N-terminal Src homology 2 domains and a C-terminal kinase domain separated by a flexible linker (Sada et al., 2001).
Syk activation has been implicated in a variety of hematopoietic cellular responses (Chu et al., 1998
; Sada et al., 2001
; Turner et al., 2000)
, and there is a growing literature supporting the role of Syk in hematological malignancies, particularly the lymphomas (Chen et al., 2008
; Feldman et al., 2008
; Rinaldi et al., 2006
; Streubel et al., 2006)
. Interestingly, we see variable patterns of p-Syk expression and response to R406 in acute lymphoid leukemia cell lines (Figure S13
and Table S5
Our demonstration of a role for Syk in the pathogenesis of myeloid malignancies is supported by the case report of the fusion of the TEL
to the Syk
gene in a patient with MDS with t(9;12)(q22;p12) (Kuno et al., 2001).
Importantly, this TEL-Syk fusion transformed the interleukin-3 dependent murine hematopoietic cell line Ba/F3 to growth factor independence (Kanie et al., 2004).
Syk mRNA transcript has been reported to be expressed in primary AML blasts (Tomasson et al., 2008)
and its expression correlated with response to treatment with gemtuzumab ozogamicin (Balaian and Ball, 2006)
. In our studies, we demonstrate that Syk protein is not only expressed but is also constitutively activated in nearly all of the samples evaluated. Determining the mechanism of activation of Syk and its critical downstream effectors in AML is under active investigation.
The screening of libraries enriched for FDA-approved compounds is an attractive strategy to repurpose drugs for alternative indications and to more rapidly test clinically relevant hypotheses in patients. However, the potency and selectivity of such drugs are often not optimal for discovered indications. Moreover, there is the desire to optimize on-target activity and minimize off-target side effects. While EGFR inhibitors have had anti-AML effects, including complete responses, these molecules do not potently inhibit Syk and are often not well tolerated at doses anticipated to inhibit Syk in vivo. Therefore, we need to identify more potent and selective Syk inhibitors for this indication. The Syk inhibitor R788 is the orally available prodrug of R406. Phase I testing of R788 has been completed with peak concentrations in excess of 10 μM, steady state concentrations of 1–3 μM, and trough concentrations of 1 μM (Elliott Grossbard, Rigel Pharmaceuticals, Personal Communication). In Phase I testing in patients with rheumatoid arthritis and heavily pretreated patients with lymphoma, the drug has been well tolerated with significant clinical activity demonstrated (Weinblatt et al., 2008 and Margaret Shipp, Personal Communication).
In our studies, the majority of primary patient AML samples responded in a clinically achievable dose range, with an IC50
below the serum trough concentration in humans. Thus, these observations suggest that inhibition of Syk can be achieved with doses in current use in clinical trials.
The last decade has seen a marked shift in the approach to cancer-related small molecule library screening performed by the pharmaceutical industry with a transition away from phenotype-based screening and a primary focus now on target-based screening. While target-based screening has been successful for known, tractable protein targets, as with any approach, it does have limitations. The majority of known oncoproteins are not considered easily druggable, and for many malignancies, driver events have not yet been identified. In addition, target-based screening is most commonly performed with ex cellulo assays and may not fully recapitulate the complexity within a cell. Alternative screening approaches, such as traditional phenotype-based and expression-based approaches, create the possibility of screening in the absence of a priori target knowledge. Moreover, expression-based screening holds promise for modulating intractable targets and can be systematized as a screening paradigm. However, the transition from compound discovery to drug development has been stymied for these cell-based screens by the challenge of identifying the protein target of the identified compound. Emerging genomic, genetic, and proteomic approaches have altered the landscape of target identification for cancer. Similarly, these approaches can transform the drug discovery process. Their integration into the compound discovery phase has already begun. Our work suggests that a cross-disciplinary integration of genome-wide expression profiling, proteomics, and high-throughput RNAi-based screening could systematize protein target identification for cell-based chemical screens. Moreover, innovations in synthetic chemistry and proteomics should further facilitate this process in the future. For example, diversity oriented synthesis small molecule collections enable incorporation of chemical handles to facilitate efficient, systematic attachment of affinity resins (Burke et al., 2003).
After affinity capture, the specific protein/compound interactions can be identified by differential isotopic labeling of amino acids as recently described (Ong et al., 2009)
In conclusion, we demonstrate that the majority of AML cell lines and primary blasts, and three in vivo AML models had a therapeutic response to Syk inhibition with effects on cell growth and differentiation. With an orally available, well-tolerated Syk inhibitor currently in clinical development for other indications, the results reported here should have immediate relevance for clinical testing of Syk inhibition in patients with AML. Furthermore, these results demonstrate the feasibility of integrating shRNA-based screening and phosphoproteomic studies to identify small molecules and their mechanism of action.