Kinase pathways are primary effectors of many targeted therapy approaches for cancer. Kinase pathways can be dysregulated by mechanisms far more diverse than chromosomal rearrangements or point mutations, which drove the initial application of kinase inhibitors to cancer. Functional screening with kinase inhibitors is one tool by which we can understand the diversity of target kinases and candidate drugs for patients before fully understanding the mechanistic rationale for kinase pathway dysregulation. By combining functional screening with genomic data, it is also possible to accelerate understanding of these mechanistic underpinnings.
Key points
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Kinases are common drug targets in myeloid malignancies.
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Kinase dysregulation occurs through a diversity of known and unknown mechanisms.
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Functional screening with kinase inhibitors can foster identification of important kinase targets in myeloid leukemia patient subsets.
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Combining functional screening with genomic data can accelerate understanding of the mechanistic etiology of kinase pathway dependence.
Introduction
Cancer therapy that is targeted to causative genetic abnormalities has achieved dramatic clinical outcomes in certain malignancy subsets. Broad application of this strategy requires a precision medicine approach in which key targets of pathway dysregulation can be rapidly assigned to specific therapeutics in individual patients. The tyrosine kinase gene family, in particular, has played a prominent role as novel targets for cancer therapy over the past several decades.
Introduction
Cancer therapy that is targeted to causative genetic abnormalities has achieved dramatic clinical outcomes in certain malignancy subsets. Broad application of this strategy requires a precision medicine approach in which key targets of pathway dysregulation can be rapidly assigned to specific therapeutics in individual patients. The tyrosine kinase gene family, in particular, has played a prominent role as novel targets for cancer therapy over the past several decades.
Kinases as gene targets in myeloid malignancy
There are several reasons that kinases and kinase inhibitors have been so broadly explored as cancer therapeutics:
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Tyrosine kinases play an integral role in numerous cellular processes as diverse as proliferation, apoptosis, differentiation, and cell motililty ; therefore, dysregulation of tyrosine kinase pathways is likely to contribute to the oncogenic process.
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There are many examples of specific kinases, both mutated and wild type, that have been directly implicated in the pathogenesis of numerous cancer subsets.
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Over the past several decades, a great deal of work has been invested in the development of large arsenals of small molecules that can potently and selectively block the activity of many specific kinases.
Effective translation of this diverse collection of small-molecule kinase inhibitors into a clinical setting requires an understanding of the individual patients and larger patient populations most likely to benefit from different kinase inhibitors. This task is more complex than it might seem, because the routes to kinase pathway dysregulation are numerous and far more complex than a simple genomic lesion in the kinase of interest. Some of these routes are listed as follows, with examples of each taken from the hematologic malignancy literature.
Chromosomal Translocation
Chronic myeloid leukemia (CML) is caused by the 9;22 chromosomal translocation, resulting in the BCR-ABL fusion gene. The same BCR-ABL gene fusion has also been implicated in adult and pediatric acute lymphoblastic leukemia (ALL), and more recently a wider diversity of tyrosine kinase gene fusions have been reported in ALL cases with similar gene expression signature, but lacking the BCR-ABL–causing chromosomal rearrangement. In addition, there are numerous rearrangements involving fusions of genes in other gene families in hematologic malignancies, and some of these have been reported to lead indirectly to kinase pathway dysregulation.
Point Mutations and Insertion/Deletions
One of the best examples of point mutations and insertion/deletions playing a prominent role in hematologic malignancies comes from the myeloproliferative neoplasms, polycythemia vera, primary myelofibrosis, and essential thrombocythemia, in which most cases were found to have point mutations in JAK2 or MPL . Subsequently, frameshift mutations in CALR were found in most JAK2/MPL wild-type cases, and these frame-shifted CALR mutants were shown to interact with and dysregulate MPL/JAK2 signaling. Importantly, this example shows that genetic lesions affecting any number of genes in nonkinase gene families can still lead to kinase pathway dysregulation through complex mechanisms.
Aberrant Expression
Aberrant expression of kinases has been reported in numerous cancers with one of the best-known examples arising from amplification of ERBB2 (HER2) in breast cancer, and examples also exist in hematologic malignancies, such as upregulation of hepatocyte growth factor and its receptor tyrosine kinase, MET, in acute myeloid leukemia (AML).
Oncorequisite Pathways
Although there are numerous examples of a kinase pathway that becomes dysregulated due to a genetic or epigenetic event, there are also emerging examples of pathways that are critical for tumor cell growth and viability without overt alterations in the sequence or expression level of these genes, and specific examples indicate these pathways can be successfully targeted. One of the best examples of this from hematologic malignancies comes from the lymphoid tumors in which the B-cell receptor, which is not mutated and naturally expressed in lymphomas at levels similar to normal B cells, has been demonstrated to be targetable with inhibitors of BCR-associated kinases such as BTK, SRC-family kinases, or SYK. Similar findings have also been made in subsets of ALL cases that similarly depend on the pre-BCR.
Microenvironment
Finally, the tumor microenvironment has been demonstrated to provide critical signals maintaining tumor cell growth and viability, and many of these signals proceed through kinase pathways. Prominent examples in the hematologic malignancies include tumor necrosis factor-alpha in myeloproliferative neoplasms, interleukin (IL)-1 in CML, and CSF1 in chronic myelomonocytic leukemia.
Functional screening as a tool to understand kinase pathway dysregulation
As detailed previously, there are numerous examples of genetic, epigenetic, and microenvironmental routes to kinase pathway dysregulation that have been discovered in hematologic malignancies. However, there is also evidence that kinase pathway dysregulation is operational in a much larger fraction of cases than can be explained by the previously described insights. In 70% to 90% of patients with AML, blast cells exhibit phosphorylation of signal transducer and activator of transcription 5, a marker for tyrosine kinase activity. Known kinase pathway alterations can only be explained in approximately 50% of cases at most. Thus, tyrosine kinases have been shown to play a role in the pathogenesis of numerous hematologic malignancies, and there remain a large number of cases that exhibit abnormal tyrosine kinase activity due to mechanisms that have yet to be uncovered. Indeed, recent Phase III clinical trial results testing the multi-kinase inhibitor, midostaurin, in patients with AML with or without mutation of FLT3 showed equivalent benefit for patients with and without FLT3 mutation, suggesting unknown kinase pathways that are targeted by midostaurin are operational in FLT3 wild-type cases.
Hence, although the previously described discoveries have led to clinical exploration of kinase inhibitors on the basis of genetic markers in certain patient subsets, scientific and clinical results have suggested that the application of kinase inhibitors may be more broadly effective than just within these genetically defined subsets. One approach for understanding of kinase pathway dysregulation in cancer is the ex vivo testing of primary patient specimens with panels of small-molecule kinase inhibitors. This approach is appealing for a variety of reasons, including speed of obtaining results (4 days or less) and success of identifying pathway dysregulation irrespective of mechanistic understanding. This latter advantage is particularly important because of the plethora of mechanisms by which kinases have been and could be found to be dysregulated in myeloid malignancies and other cancer subtypes. As detailed later in this article, there are many examples in which kinase inhibitor functional screening has been used to identify novel targets in patients with myeloid malignancy and in some instances, integration of these functional screening data with genomic information has led to new understanding of the mechanistic etiology of kinase pathway dependence.
Kinase Inhibitor Screening to Identify Pathway Dependence in Individual Patients
Functional screening with kinase inhibitors can be useful to identify candidate drugs that can be applied on an individualized basis ( Fig. 1 ). One early example of this came in the form of a patient with systemic mastocytosis with associated monocytic leukemia. In this case, screening with panels of small interfering RNAs (siRNAs) and kinase inhibitors revealed dramatic sensitivity of circulating neoplastic monocytes to silencing or inhibition of JAK2. This led to sequencing of JAK2 and upstream receptors and the identification of a novel insertional mutation in the thrombopoietin receptor, MPL, which was confirmed as a transforming variant of MPL that caused dysregulation of JAK2 signaling. This patient was eligible for a clinical trial with the broad-spectrum kinase inhibitor, midostaurin, which was confirmed to inhibit viability of this patient’s cells and to block JAK2 signaling downstream of this transforming variant of MPL. The patient exhibited a dramatic clinical response to midostaurin. Interestingly, this variant of MPL has never been reported in any other patient, confirming the capacity of functional screening to identify actionable kinase pathway targets, even in circumstances of exceedingly rare, unknown genetic lesions.
Cohort-Based Kinase Inhibitor Screening
Functional screening with kinase inhibitors also has been used more broadly to survey larger cohorts of patients, thereby understanding both individual patient specimen responses as well as larger patterns in overall patient cohorts ( Fig. 2 ). In 2 of the earliest instances, 2 independent screening platforms applied to 2 distinct patient populations revealed strikingly similar patterns of drug sensitivity, and both studies successfully correlated ex vivo drug response data with clinical responses to targeted therapies.
Common Pathways/Drugs for Disease Subsets
In addition to understanding the diversity of responses across cohorts of patient specimens, kinase inhibitor screening has also been used to understand and confirm common patterns of pathway dependence in defined diagnostic hem malignancy subsets (see Fig. 2 ). Examples include the confirmation that the multi-kinase/ABL/SRC/FLT3 inhibitor, ponatinib, is indeed effective against patients with AML with FLT3-ITD, and the PI3K-delta inhibitor, idelalisib, is broadly effective in chronic lymphocytic leukemia.
Drug Repurposing
In addition, kinase inhibitor screening has, in some cases, led to surprising discovery that certain drugs can be repurposed for very specific clinical situations. One example of this comes with CML cases with mutation of T315I in the kinase domain of BCR-ABL. Before this study, the only inhibitor approved by the Food and Drug Administration with activity against BCR-ABL T315I was ponatinib; however, kinase inhibitor screening of CML patient samples revealed that cases with T315I were preferentially sensitivity to the kinase inhibitor, axitinib. Subsequent studies revealed that axitinib is preferentially active against the T315I variant of BCR-ABL relative to native BCR-ABL, and these studies have suggested that axitinib, alone or in combination with other ABL kinase inhibitors, may be an effective therapeutic option for patients with T315I-positive CML. Another example of drug repurposing has come from recent studies of AML with biallelic CEBPA mutation in which functional sensitivity to JAK kinase inhibitors was correlated with a JAK kinase gene expression signature, as well as mutation of JAK pathway nodes.
Kinase Inhibitor Screening to Refine Diagnostic and Therapeutic Markers
In addition to understanding of specific kinase inhibitor sensitivity in individual patients and the diversity of responses across populations, kinase inhibitor screening has also been used identify consistent and heretofore unknown kinase pathway dysregulation within distinct clinical patient subsets ( Fig. 3 ). One prominent example comes with the discovery of JAK and SRC-family pathway sensitivity in Philadelphia-negative neutrophilic leukemia cases downstream of somatic mutation of CSF3R (G-CSFR). The 2001 World Health Organization’s guidelines for diagnostic subsets of myeloid malignancies restricted classification of CML only to cases exhibiting the 9;22, Philadelphia chromosome rearrangement (Ph). A small number of patients exhibited disease that was clinically similar to CML, but was Ph-negative. For some time, the genetic etiology of these Ph-negative neutrophilic leukemias (chronic neutrophilic leukemia [CNL]; atypical CML [aCML]) remained unknown. Kinase inhibitor screening of patients with CNL revealed sensitivity to JAK and/or SRC-family kinase inhibitors, which corresponded to novel mutations in CSF3R, identified by genomic analyses of these cases. The CSF3R mutations segregated into 2 classes: membrane proximal mutations, which corresponded to JAK inhibitor sensitivity, and cytoplasmic truncating mutations, similar to those observed in a subset of patients with congenital neutropenia, which corresponded to sensitivity to SRC-family kinase inhibitors. A syngeneic, bone marrow transplant model of CSF3R mutation recapitulated CNL disease phenotype, which was mitigated with JAK inhibitor treatment. These findings ultimately led to the discovery that CSF3R mutations are highly prevalent in patients with CNL and are observed with lower frequency in patients with aCML. In the recent update to World Health Organization diagnostic guidelines, CSF3R mutation has been included as a diagnostic feature of CNL. Functional screening platforms also can be extensible to other families of small-molecule inhibitors to make similar types of discoveries, such as the recent identification of a HOX gene expression signature as a marker of sensitivity to BH3 mimetics.
Related posts:
Tyrosine Kinase Inhibitor Treatment for Newly Diagnosed Chronic Myeloid Leukemia
Mechanisms of Resistance to ABL Kinase Inhibition in Chronic Myeloid Leukemia and the Development of Next Generation ABL Kinase Inhibitors
The Development and Use of Janus Kinase 2 Inhibitors for the Treatment of Myeloproliferative Neoplasms
Mechanisms of Resistance to JAK2 Inhibitors in Myeloproliferative Neoplasms
The Development of FLT3 Inhibitors in Acute Myeloid Leukemia
Identification and Targeting of Kinase Alterations in Histiocytic Neoplasms
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