SU11248 shows pre-clinical efficacy in a variety of cancer models
~ An innovative phase I trial for targeted cancer therapy~
Class presentation: March 8th, 2005.
Paper presented: O'Farrell et al.,
Clinical Cancer Research. 9:5465-5476., 2003.
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I. Angiogenesis and Cancer
Formation of new vasculature within adults is accomplished through a number of coordinated processes. Briefly, current vessel must be destabilized and matrix degradation must occur to allow endothelial cell migration. Endothelial cells then proliferate and form tubelike structures. Pericytes are then recruited to the abluminal surface of the endothelium. The vessel is then mature and blood flow is established. This process is reviewed in (1) with excellent diagrams.
In cancer, anti-angiogenic agents have been shown moderately effective in preventing recurrence of disease in an adjuvant setting (ASCO). When a solid tumor is 1-2 mm in size, it is able to obtain nutrients by passive diffusion. Once the tumor grows larger, it must develop its own blood supply. Tumor derived angiogenesis mimics normal angiogenesis, but ultrastructurally can be quite abnormal (1). Many tumor derived vessels lack pericytes, and phenotypically are dilated and tortuous in nature. The vessel wall itself is a chimera of tumor cells and endothelial cells. Despite these structural abnormalities, they are able to provide nutrients to the growing tumor.
Vascular endothelial growth factor, or VEGF, is an important growth factor required for the process of angiogenesis. Therefore, it is thought that blocking VEGF signaling may prevent tumor vascularization.
I. SU11248 is Anti-angiogenic. Study by Osusky et al. (2)
SU11248 is a compound licensed by SUGEN, South San Francisco, CA. It is a potent and selective inhibitor of the kinase insert domain containing the receptor (KDR/FLK-1) for VEGF and PDGFRb with an IC50 >10µM (3). Refer to Osusky et al. for published data, but in a brief summation they incorporated multiple in vitro and in vivo angiogenesis models to assess the effect of SU11248 on the formation of new blood vessels. They demonstrated that SU11248 impairs endothelial cell migration using Boyden chamber assay and a simulated wound assay. They then showed that SU11248 drastically decreased the formation of microcentric junctions in a matrigel assay. For in vivo investigation, they used a tumor window model and a Lewis Lung Carcinoma models in C57BL/6J mice. Both assays confirmed that administration of SU11248 attenuated microvascularization and metastasis. In conclusion, they suggest that VEGF and tyrosine kinase inhibitors may have clinical implications such as preventing tumor recurrence or metastasis after surgery, radiotherapy, or chemotherapy.
II. FLT3 biology and relevance in leukemias
FLT3 is a tyrosine kinase receptor expressed in a variety of myeloid and B-lymphoid lineage cells. The expression of FLT3 is restricted to early bone marrow progenitor cells expressing CD34+. See Figure below (from Wodner-Filipowicz, News Physiol Science, 2003, Ref 4) for a diagram of FLT3 biological activities.
Because FLT3 and its ligand FL is involved in maintenance, expansion, and differentiation of hematopoietic stem cells, it is not surprising that it is expressed at high levels in a variety of hematological malignancies. Studies from FLT3 knockout mice illustrate that though the adults develop normally, they do exhibit deficiencies in primitive B-lymphoid populations (5) .
Mutations in the FLT3 receptor have been detected in ~30% of patients with acute myelogenous leukemia (AML), this is the single most commonly mutated gene in AML (5)(6) These mutations may play a role in the survival or proliferation of leukemic blasts. Although FLT3 mutations may play a role in the disease process, FLT3 is not sufficient to cause AML, an additional mutation must be acquired to present with full disease(7). See figure from Gilliland and Griffin, Blood, 2002 for a diagram of the "two-hit" hypothesis.
III. FLT3 mutations
Two groups of FLT3 mutations are found in subsets of AML patients. FLT3 contains a Juxtamembrane domain (JM) that functions to inhibit the kinase activity. Mutations in this domain, also called Internal tandem duplications (ITD) cause a helical conformation that distorts the small lobe of the kinase domain and blocks the JM domain from performing its "autoinhibitory" function. See below for a diagram from Gilliland and Griffen, Blood (7) which delineates the mutations.
FLT3 activation occurs when phosphorylation causes a conformational change. Another category of mutations is in the active loop. The active loop folds into the ATP and substrate binding pocket blocking kinase activation. If mutated, the active loop folds out of the active site and allows continuous access to ATP and substrate. Ribbon diagrams demonstrating these mutations can be found in Naoe and Kiyoi, Cell Mol Life Sci (5).
IV. SU11248 inhibits FLT3 in vivo
The FLT3 ITD mutation is found in 25-30% of AML patients (7). Additionally it is the single most significant poor prognosis factor in AML in several independent studies (8) (9). Because of the significance of the FLT3-ITD mutation, O'Farrell et al. (10) They used multiple in vitro assays to demonstrate SU11248 inhibits FLT3 kinase and induces apoptosis in wild type as well as FLT3-ITD and activation loop mutants. They also used a subcutaneous tumor xenograft model to show that SU11248 can induce regression of tumors derived from implanted MV4;11 cells which express the FLT3-ITD mutant. In this study they show dramatic tumor destruction with visual disappearance after 4 days in all of the ten mice used. After withdrawal of SU11248, six of the ten mice exhibited tumor regrowth. O'Farrell et al. then investigated the effects of SU11248 in a more physiologically relevant leukemia model: a bone marrow engraftment model. They implanted the FLT3-ITD expressing cells in NOD-SCID mice after endogenous bone marrow ablation. After administering daily SU11248 or control they watched the mice for clinical signs of "leukemia". Mice receiving the highest dose SU11248 were disease free a mean of 83 days compared to control mice who dies at a mean of 41 days. This data gives more support to the hypothesis that SU11248 could oppose FLT3 dependant AML in humans.
V. Targeted therapy in human trials: An Innovative Phase I clinical study demonstrates an inhibition of FLT3 phosphorylation by SU11248 in AML patients
O'Farrell et al., Clinical Cancer Research. 9:5465-5476., 2003.
Pre-clinical data outlined above suggests that SU11248 may be useful in a variety of clinical cancer settings. The question remains how to demonstrate that the drug is hitting its target in human trials. These authors took a unique approach to a phase I trial by measuring FLT3 activity in the blood of AML patients after a single dose of the drug.
The intent of the study was not only to determine the pharmacodynamics and pharmacokinetics of SU11248, but also to show levels of FLT3 activity and its downstream targets.
This study enrolled 29 patients, 93% having received at least one prior therapy. Each patient received a single dose of SU1128 ranging 50-350mg. Adverse events were reported in 31%, with nausea being the only AE over 10% in frequency. Most AE were reported only at the higher doses. Results from the study will be summarized below. Please refer to the manuscript for the specific details.
Patients exhibited a dose-proportional increase in both Cmax (highest plasma concentration) and AUC (total exposure). Across all dose levels, the Tmax (time of maximal plasma concentration) was generally observed at 4-8 hrs. The calculated half-life was 44 +/- 18 hours.
Patients were genotyped for FLT3 to determine if they carry an ITD or activation loop mutations. 10.3% had an ITD mutation, 6.8% had activation loop mutations and the rest were wild type FLT3. FLT3 phosphorylation was present in 73% of the patients pre-treatment. After receiving SU11248, 77% of the patients receiving >200mg had a strong inhibition of FLT3. Only 25% of the patients receiving <200mg showed a response in FLT3, of those all were FLT3 mutant, suggesting the mutant FLT3 was increased sensitivity to SU11248.
Data was plotted to assess if the PK/PD correlated with activity. Generally, the plasma Cmax was lower in patients with a weak response versus a strong response. A mean of 100ng/ml appeared to be required for strong activity.
Downstream mediators of FLT3 signaling were assayed for phosphorylation status. Stat5 is known to be activated by proteins associated with leukemogenesis. Stat5 is often found in activated form in AML blasts. Pre-dose Stat5 phosphorylation was seen in 89% of patients in the study. SU11248 treatment was associated with decreased Stat5 in the majority of patients. Two of the WT FLT3 patients who exhibited strong FLT3 inhibition did not show Stat5 inhibition at any timepoint.
FLT3 also leads to phosphorylation and activation of ERK1/2. Pre-dose ERK phosphorylation was observed in 69% of patients. This phosphorylation was decreased in 80% of the patients after SU11248 administration. No obvious correlation was apparent between ERK1/2 inhibition, FLT3 modulation, FLT3 genotype, or PK.
This study provided direct evidence of the biological activity of SU11248 on FLT3 inhibition in AML patients. Although evidence of ERK and Stat5 modulation was apparent, it was difficult to interpret and draw a correlation to SU11248 activity. Additional studies and more patients will be needed to determine if ERK1/2 is a possible biomarker for SU11248 activity. Therefore, direct measurement of FLT3 phosphorylation was the most valuable correlative information obtained in this study.
Proof of inhibition of the target in a clinical setting provides valuable information and guides additional clinical development of SU11248, which is in phase I and II clinical trials.
VI. References
1. Papetti and Herman, Mechanisms of Normal and tumor derived angiogenesis.
American Journal of Physiology, 282: C947-C970, 2002.
2. Osusky L., Hallahan D E., Fu A, Fei Y., Shyr Yu., Geng L. The receptor tyrosine kinase inhibitor SU11248 impedes endothelial cell migration and blood vessel formation in vivo, but has little effect on existing tumor vessels. Angiogenesis, 7: 225-233, 2004.
3. Mendel DB., Laird AD., Xin X. et al., In vivo antitumor activity of SU11248, a novel tyrosine kinase inhibitor targeting vascular endothelial growth factor and platelet derived growth factor receptors: Determination of a pharmacokinetic/pharmacodynamic relationship. Clin Cancer Research, 9:327-337, 2003.
4. Wodnar-Filipowicz A., Flt3 ligand: role in the control of hematopoietic and immune functions of the bone marrow. News Physiol Science, 18:247-251, 2003.
5. Naoe T. and Kiyoi H. Normal and Oncogenic FLT3 Cell Mol Life Sci, 61: 2932-2938, 2004.
6. Nakao M., Yokota S., Iwai T., Kaneko H., Horike S., Kashima K., Sonoda Y., Fujimoto T., and Misawa S., Internal tandem duplication of the FLT3 gene found in acute myeloid leukemia. . Leukemia, 10: 1911-1918, 1996.
7. Gilliland and Griffin, The roles of FLT3 in hematopoiesis and leukemia. . Blood, 100 : 1532-1542, 2002.
8. Abu-Duhier FM., Goodeve AC, Wilson GA., et al. FLT3 internal tandem duplication mutations in adult acute myeloid leukemia define a high-risk group. British Journal of Haematology. 111: 190-195, 2000.
9. Meshinchi S., Woods WG., Stirewalt DL., et al. Prevalence and prognostic significance of Flt3 internal tandem duplication in pediatric acute myeloid leukemia. Blood 97: 89-94, 2001.
10. O'Farrell A, Abrams TJ, Yuen HA, Ngai TJ, Louie SG., Yee K., Wong LM., Hong W., Lee LB, Town A., Smolich BD., Manning WC., Murray LJ., Heinrich MC., and JM Cherrington. SU11248 is a novel FLT3 tyrosine kinase inhibitor with potent activity in vitro and in vivo. Blood. 101: 3597-3605, 2003
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11. O'Farrell A, Foran JM., Fiedler W., Serve H., Paquette RI., Cooper M., Yuen HA., Louie SG., Kim H., Nicholas S., Heinrich MC., Berdel WE., Bello C., Jacobs M., Seicalla P., Manning WC., Kelsey S., and Cherrington JM. An Innovative Phase I clinical study demonstrates inhibition of FLT3 phosphorylation by SU11248 in acute myeloid leukemia patients. Clinical Cancer Research, 9: 5465-5476. 2003.
Presented by Bethany Merenick (bethany.merenick@dartmouth.edu)