- Email: [email protected]
Can FLT3 inhibitors overcome resistance in AML?
Best Practice & Research Clinical Haematology Vol. 21, No. 1, pp. 13–20, 2008 doi:10.1016/j.beha.2007.11.003 available online at http://www.sciencedirect.com
2 Can FLT3 inhibitors overcome resistance in AML? Winnie F. Tam
PhD
D. Gary Gilliland *
PhD, MD Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA Dana-Farber Cancer Institute, Howard Hughes Medical Institute, Harvard Medical School, Boston, MA, USA
The identification of FLT3 mutations across a range of the cytogenetic subgroups of AML has opened up the possibility of a targeted therapeutic approach with broad applicability. Four agents are currently in clinical trials, at least 3 of which have both sufficient activity against AML and sufficiently acceptable toxicity profiles to support continued efforts to refine their inclusion into therapeutic regimens for AML. Better understanding of the genetics of inherent and acquired resistance is needed to guide development of second-generation agents. Optimizing the integration of FLT3 inhibitor therapy with chemotherapy has the potential both to decrease toxicity and improve response. Key words: FLT3 inhibitors; resistance; staurosporine; midostaurin; PKC412; tandutinib; MLN 518; lestaurtinib; CEP701; SU11248; AML.
INTRODUCTION FLT3 is a class III receptor tyrosine kinase (RTK) expressed on hematopoietic progenitors. It is normally inactive when not bound to its ligand, and is autoinhibited by a juxtamembrane domain that folds structurally into its catalytic site. FLT3 is mutated in about 30% of the patiens with acute myelogenous leukemia (AML); these are lossof-function mutations in the autoinhibitory domain in the form of internal tandem duplications near the juxtamembrane region, or point mutations in the activation loop. Either type of mutation results in constitutive activation of the FLT3 tyrosine kinase in the absence of ligand.1
* Department of Medicine, Brigham and Women’s Hospital, 1 Blackfan Circle, Room 5.0210, Boston, MA 02115, USA. Tel.: þ1 (617) 355 9092; Fax: þ1 (617) 355 9093. E-mail address: [email protected] (D. Gary Gilliland). 1521-6926/$ - see front matter ª 2007 Published by Elsevier Ltd.
14 W. F. Tam and D. Gary Gilliland
FLT3 internal tandem duplications (ITDs) have been identified in virtually every subtype of leukemia including the different cytogenetic risk groups.2 Certain favorable risk groups have a relatively high proportion, including almost 40% in patients with acute promyelocytic leukemia. The mutation is also common in several of the intermediate risk groups, including those with normal cytogenetics. It is not rare in patients that have adverse risk cytogenetics, but importantly, in any of the cytogenetic risk groups, the FLT3 mutation appears to confer a poor prognosis. For example, nearly all poor-risk, ITD-positive patients will relapse within a year, whereas poor-risk ITD-negative patients have a relapse-free survival of about 30% at 1 year. FLT3 positive patients are thus a prognostic group in which molecularly targeted therapy could have a significant therapeutic impact. FLT3 INHIBITORS CURRENTLY IN CLINICAL TRIALS Four different FLT3 inhibitors have reached clinical trials in AML: MLN-518/tandutinib, a quinazolone compound from Millennium;, PKC-412/midostaruin, a benzoyl staurosproine from Novartis, CEP-701/lestaurtinib, an indolocarbazole from Cephalon, and an indolinone Su-11248, initially developed by SuGen, now at Pfizer, which has been associated with cardiotoxic effects3, and has primarily been evaluated in the context of solid tumors.4,5 PKC-412 from Novartis is benzoyl staurosporine.6 The indolocarbazole derivatives from Cephalon were developed in collaboration with Donald Small and colleagues at Hopkins.7 Millennium’s MLN-518 also known as tandutinib, has a 50% inhibitory concentration (IC50) for FLT3, PDGF receptor, and c-KIT of approximately 30 nanomolar (nM), is metabolically stable, and is orally bioavailable. Research groups have shown that tandutinib is efficacious in murine models of FLT3 ITD-induced disease.8 In work on tandutinib, there is in vitro inhibition of FLT3 in cell culture systems with IC50s for inhibition of autophosphorylation of about 30 nM. Preparations from patients with FLT ITDs treated with the drug at doses of 525 mg bid show reduction in phospho-FLT3 content within several hours after initiation of therapy that persists for several days. These data were reported as part of a phase I, open-label trial in 40 patients with relapsed or refractory AML or high risk myelodysplastic syndrome (MDS). In this study, FLT3-ITD mutations were not required as a criterion for enrollment. A single agent, median tolerated dose (MTD) of 525 mg bid was established. The dose-limiting toxicity was reversible; muscle weakness and pharmacodynamics were consistent with an in vivo IC90 of about 350 nM.9 A phase II trial also conducted with this group, using the MTD from the prior study of 525 mg bid in 25 FLT3-ITDpositive patients, showed more than a 50% decrease in bone marrow blasts in 11/25, and 1 patient had a partial CR.9,10 Several patients in this study had dramatic reductions in blast percentage in the peripheral blood, for example from 39,000/mL down to 400/mL or from 12,000/mL down to nearly 0/mL. These are impressive responses in the peripheral blood. The responses in the bone marrow were less dramatic, exhibiting a reduction in blast percentage from 91% to 62% and 80% to 15% on day 28. Other FLT3 inhibitors as well have showed similar responses.3,11,12 DeAngelo and colleagues have followed up these initial results with a phase I/II study of MLN518 plus standard induction chemotherapy in newly diagnosed AML patients. The schema was standard 3 þ 7, daunoribicin and cytosine arabinoside induction with concomitant MLN518 given days 0 to 14 with an option for a second induction if there was persistent leukemia from the bone marrow aspirate and biopsy at day 14. The overall response rate in this study was 70% with 21 of 30 patients achieving a CR.9 The MLN518 was quite well tolerated in combination with the
Can FLT3 inhibitors overcome resistance in AML? 15
standard induction, demonstrating the usual nausea, diarrhea, and vomiting as adverse events. Neutropenia, thrombocytopenia, and a prolonged QTc were the most common grade 3 adverse events and occurred in about 10% of the patients. This suggests that this drug has clinical activity with chemotherapy and that further studies may be warranted to characterize it in randomized trials. PKC412, or midostaurin was developed by Novartis, and is N-benzoylstaurosporine. It was originally developed as a vascular endothelial growth factor (VEGF) and protein kinase C (PKC) inhibitor as the title indicates. A phase I trial was completed, and PKC412 was found to be a FLT3 inhibitor with an IC50 of less than 10 nM. PKC412 also specifically inhibits the growth of leukemic cell lines that are made factor-independent by transfection of the constitutively activating FLT3 mutations, either ITD or D835Y point mutations. It has exhibited activity in FLT3-ITD-induced disease in murine bone marrow transplantation models.1,13 A phase II trial with PKC412 in AML conducted by Richard Stone and colleagues on 25 patients demonstrated greater than 50% reduction in bone marrow blasts in 25% of patients. Two patients had less fewer than 5% blasts, 1 at day 28 and 1 at day 60. Peripheral blood blasts were reduced by more than 70%, including 3 patients who showed a reduction in peripheral blood blast percentage from 110,000/mL to 0, 61,000/mL to 0 and 46,000/mL to 0 at days 29, 57, and 51. The peripheral blood response was again more impressive than the bone marrow response. Seven patients (35%) appeared to have clinical benefit.14 The phase II trial led to a phase IB study of PKC412 in combination with daunorubicin and cytarabine in newly diagnosed patients. The total CR rate was 71% and it was significantly higher in those patients who had FLT-ITD mutations. Eleven of 12 patients went into CR, suggesting that there may be added benefit in patients that are mutant as opposed to wild type. Among patients who responded, 4 subsequently relapsed. These occurred relatively rapidly at 7, 7, 10, and 15 months respectively, but 7 of 11 remain in CR with follow-up of 3 to 15 months.15 It is problematic that the frequent appearance of rapid relapse in responding patients has also emerged in the treatment of chronic myelogenous leukemia (CML) blast crisis with imatinib. The Cancer and Leukemia Group B (CALGB), Southwest Oncology Group (SWOG), and Eastern Cooperative Oncology Group (ECOG) plan to enter into an intergroup study in which the use of PKC412 will be randomized with patients treated with induction chemotherapy with standard 3 þ 7. Patients will be under the age of 60 years and must have mutated FLT3. The study design requires access to the screening of a large number of patients, and the protocol is still in development. CEP701, or lestaurtinib developed by Cephalon, is an indolocarbazole derivative. It has good oral bioavailability. It has a long half-life in murine model systems, and Dr. Small and his colleagues at Johns Hopkins showed in murine model systems that it is a potent inhibitor of FLT3. Reduction in the phospho-FLT3 signal can be seen several hours after initiation of therapy and then recrudescence of the signal at about 12 hours. In a model system using a murine cell line containing the ITD mutation injected into syngeneic recipients, the drug is effective at prolonging survival compared with placebo controls.7,16 Based on these preclinical platforms, a randomized openlabel study was conducted by Dr. Small and colleagues. This study demonstrated that clinical response appears to correlate well with FLT3 inhibition. The trial design took AML patients in first relapse with FLT3 mutations, randomized them to receive either chemotherapy alone or chemotherapy plus CEP701, days 7 through 42. Patients in the control arm were eligible for crossover after assessing initial response. Chemotherapy was based on remission duration. Patients with a remission of 1 to 6 months were treated with mitoxantrone, etoposide and cytarabine. Those with a remission of
16 W. F. Tam and D. Gary Gilliland
6 to 24 months were treated with high dose cytarabine.16 In the 22 patients treated in the chemotherapy plus CEP701 arm, 10 had either full or partial CR (5 and 5), and 9 had no response. Among those 22 patients in the chemotherapy arm, 6 had either full or partial CR (3 and 3) and 15 had no response, suggesting a trend toward efficacy in the chemotherapy þ CEP701 arm. However, in vitro, regardless of mutational status of FLT3, 8 patients who were full or partial responders could also show pharmacodynamically that the target was inhibited in vivo, whereas patients that did not have in vitro sensitivity or in which FLT3 could not be inhibited in vivo were less likely to have responses. This suggests that response correlates with the degree of FLT3 inhibition and that pharmacodynamic monitoring or pretrial assessment may be of value in predicting which patients will respond in future trials. CEP701 also shows cytotoxic activity in a large proportion of AML patients that have FLT3 mutations.17 Sustained plasma levels can be obtained in about 60% of patients with 80 mg bid. The plasma FLT3 inhibitory activity combined with in vitro susceptibility to CEP701 shows a high correlation with clinical response in these trials, suggesting that this may offer benefit to those patients that have FLT3 activating mutations. This is also being tested in a trial that was initiated in Europe. None of the four FLT-3 inhibitors currently in clinical trials offers obvious advantages over the others in terms of response or side effect profile. Most of these appear to be very well tolerated with modest toxicities; using pharmacodynamic estimates, they all appear to inhibit the pharmacologic target. All appear to be active in treating patients with FLT3 mutations, but the most impressive clinical responses have been a reduction of peripheral blood blast percentage which has been more impressive than the reduction in bone marrow blast percentage. Patients that overexpress the wild type also respond, and resistance develops rapidly in those patients who do respond. GUIDING THE FUTURE DEVELOPMENT OF FLT3 INHIBITORS: OVERCOMING RESISTANCE Understanding the background of the activity of FLT3 inhibitors, several topics should guide the next steps of their development. These include undestanding why peripheral blood responses are consistently more impressive than bone marrow responses; the reason why FLT3 inhibitors do not seem to work as effectively as imatinib for CML blast crisis, and the basis for rapid development of resistance. Increasing potency of FLT3 inhibitors may be of value if it can be tolerated without toxicity in patients with AML, and there are several companies that are working actively to develop more potent inhibitors. One potential explanation for the bone marrow versus peripheral blood responses may be microenvironmental resistance to FLT3 inhibitors. Growth factors or stromal cells in the bone marrow may rescue leukemic cells from FLT3 inhibition. If that is so, then in the peripheral blood where cells do not have stromal support, leukemic cells would be highly susceptible to inhibition with FLT3 inhibitors. In the bone marrow, where leukemic cells can be protected by growth factor support, they would be less susceptible to induction of cell death. This model allows consideration of potential solutions, such as trying to mobilize bone marrow blasts in advance of treatment with FLT3 inhibitors, using chemotherapy or growth factors for mobilization, or more elaborate strategies like CXCR4 antagonists or stromal derived factor-1 (SDF1) antagonists that would mobilize blasts into the peripheral blood. The reason why FLT3 inhibitors are not apparently as efficacious in AML as imatinib in the treatment of CML in blast crisis is problematic. In BCR-ABL positive disease, the
Can FLT3 inhibitors overcome resistance in AML? 17
stable phase is associated with expression of BCR-ABL in all cells. The cells that progress to blast crisis may be associated with additional mutations. But nonetheless, in blast crisis, every malignant clonal cell will have BCR-ABL present and these cells are susceptible to inhibition with imatinib, which can induce at least transient CRs until resistance develops. (Figure 1) The FLT3-ITDs mutations in AML are different because, in at least some patients, they may be acquired late in the disease. The status of FLT3ITD in paired diagnosis and relapse samples is an example. Some patients are wild type at diagnosis and at time of relapse; some patients start as wild types and acquire ITD at the time of relapse; some patients have the ITD initially and are wild type at time of relapse; and then of course some patients will have the ITD at diagnosis and at relapse. The ITD can therefore be present at the time of initial therapy but relapse with the clone of cells that lacks the FLT3-ITD2,18–20, differentiating it from BCR-ABL disease. If the FLT3 mutation is present early in disease pathogenesis then the inhibitor could Stable Phase
BCRABL
BCR -ABL BCR -ABL BCR -ABL BCR -ABL
Blast crisis Additional mutations
BCR -ABL BCR -ABL BCR -ABL
imatinib
CR
BCR -ABL
Figure 1. CML in blast crisis can be treated with imatinib because all cells harbor the BCR-ABL mutations. In contrast, FLT3-ITD may be acquired late in disease progression such that not all leukemic blasts have the FLT3-ITD mutations. Thus, treatment with FLT3-ITD inhibitors is not capable of eradicating all leukemia cells resulting in resistance or relapse of disease. CML malignant clones express BCR-ABL in early disease stage and later in blast crisis. For this reason imatinib may be effective in refractory disease because these clones are sensitive to imatinib. FLT3 inhibitors may not be as efficacious in AML as imatinib for treatment of CML blast crisis because the FLT3-ITD mutations can occur at any stage of the disease (see illustration below).
18 W. F. Tam and D. Gary Gilliland
potentially eradicate the leukemic clone leading to CR. However, if FLT3 is acquired as a secondary event in disease pathogenesis, then not all leukemia cells will have the FLT3-ITD. In that situation, even if the inhibitor is effective, the patient is left with a resistant clone that is FLT3-ITD negative and could lead to early relapse. The use of FLT3 inhibitors is unlikely to circumvent this problem, and so combination therapy will be important. In those AML patients who do respond to FLT3 inhibitors, resistance develops rapidly due to increased drug clearance, inherent resistance and the acquisition of resistance mutations analogous to imatinib resistance BCR-ABL mutations. Inherent resistance can be documented with tandutinib in the form of variable sensitivity of the FLT3 activation loop mutations to this particular inhibitor.21 The ITD mutation has an IC50 in this particular cellular context of about 0.550 mM whereas the D835Ys or D835Vs are very highly resistant, with IC50 > 10 mM. These results indicate that it is necessary to think in advance about genotyping patients to predict response to therapy, especially therapy based on FLT3 inhibition. It is also necessary to look at all the activation loop alleles in terms of their sensitivity or spectrum of sensitivity to the inhibitors that are being used in clinical trials. Jahn Cools has examined acquired resistance to FLT3 inhibition using PKC412 resistance mutations and has identified several alleles that confer resistance to PKC412.22 Figure 2 illustrates several of these interacting with the PKC412 molecule where the G697 or N676 mutations can presumably displace PKC from the catalytic pocket. These same two mutations also confer a high degree of resistance to the other three inhibitors discussed above. These potential resistance alleles need to be considered in advance. Thomas Fisher and colleagues, in a clinical trial using PKC412 to treat AML, have identified N676D mutations as conferring clinical resistance.23 Second generation drugs can be proactively developed that are more potent and that will overcome these resistance point mutations if this problem is anticipated. As described above, in vitro analysis shows that there are mutations that confer
Figure 2. Acquired resistance: modeling PKC412 resistance mutations on FLT3 structure. In this model acquired mutations G697 and N676 can displace the inhibitor PKC412 from blocking the activating site, and in this case, the receptor remains constitutively active. The stick figure in green is the PKC412 molecule.22
Can FLT3 inhibitors overcome resistance in AML? 19
a high degree of resistance to PKC412, and 2 of these mutations also confer high resistance to other FLT3 inhibitors, including CEP701 and tandutinib (MLN518). Compounds that will override these resistance alleles should be developed. The next step in developing the use of FLT3 inhibitors to overcome inherent resistance in AML is to use combination therapies. This includes working out the optimal sequencing of chemotherapy and FLT3 inhibitors. For example, Dr. Small and colleagues at Hopkins have developed some in vitro data that suggests that giving chemotherapy first may have some cytotoxic advantage24, though extrapolation into human patients may be problematic. In Dr. Stone’s phase IB trial of PKC412, discussed above15, significant toxicity was encountered when PKC412 was given concurrently with induction chemotherapy. As a result, the study drug is now being sequenced in after the chemotherapy. Along with decreasing toxicity of the combined regimen, this approach may also mobilize the blasts from the microenvironmental protection and improve marrow response. SUMMARY AND CONCLUSIONS The identification of FLT3 mutations across a range of the cytogenetic subgroups of AML has opened up the possibility of a targeted therapeutic approach with broad applicability. However, to guide development of second generation agents, better understanding is needed of the genetics of inherent and acquired resistance, the microenvironmental niche and other factors that appear to shelter blasts in the marrow from FLT3 inhibition in the peripheral blood, and the processes of rapid relapse after initial response. Optimizing the integration of FLT3 inhibitor therapy with chemotherapy has the potential both to decrease toxicity and improve response. REFERENCES 1. Kelly LM, Liu Q, Kutok JL et al. FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myeloproliferative disease in a murine bone marrow transplant model. Blood 2002; 99: 310–318. *2. Kottaridis PD, Gale RE, Frew ME et al. The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy: analysis of 854 patients from the United Kingdom Medical Research Council AML 10 and 12 trials. Blood 2001; 98: 1752–1759. *3. Fiedler W, Serve H, Dohner H et al. A phase 1 study of SU11248 in the treatment of patients with refractory or resistant acute myeloid leukemia (AML) or not amenable to conventional therapy for the disease. Blood 2005; 105: 986–993. 4. Motzer RJ, Rini BI, Bukowski RM et al. Sunitinib in patients with metastatic renal cell carcinoma. The Journal of the American Medical Association 2006; 295: 2516–2524. 5. Motzer RJ, Hoosen S, Bello CL et al. Sunitinib malate for the treatment of solid tumours: a review of current clinical data. Expert Opinion on Investigational Drugs 2006; 15: 553–561. 6. Fabbro D, Ruetz S, Bodis S et al. PKC412–a protein kinase inhibitor with a broad therapeutic potential. Anti-Cancer Drug Design 2000; 15: 17–28. *7. Levis M, Allebach J, Tse KF et al. A FLT3-targeted tyrosine kinase inhibitor is cytotoxic to leukemia cells in vitro and in vivo. Blood 2002; 99: 3885–3891. *8. Kelly LM, Yu JC, Boulton CL et al. CT53518, a novel selective FLT3 antagonist for the treatment of acute myelogenous leukemia (AML). Cancer Cell 2002; 1: 421–432. 9. DeAngelo DJ, Amrein PC, Kovacsocics TJ et al. Phase 1/2 study of tandutinib (MLN518) plus standard induction chemotherapy in newly diagnosed acute myelogenous leukemia (AML). Blood 2006; 108: 51a. [abstract 158].
20 W. F. Tam and D. Gary Gilliland 10. De Angelo DJ, Stone R, Heaney ML et al. Phase II ealuation of the tyrosine kinase inhibitor MLN518 in patients with acute myeloid leukemia (AML) bearing a FLT3 internal tandem duplication (ITD) mutation. Blood 2004; 104: 496a. [abstract 1792]. 11. Stone RM, De AJ, Galinsky I et al. PKC 412 FLT3 inhibitor therapy in AML: results of a phase II trial. Annals of Hematology 2004; 83(Suppl. 1): S89–S90. 12. Knapper S, Burnett AK, Littlewood Tet al. A phase 2 trial of the FLT3 inhibitor lestaurtinib (CEP701) as first-line treatment for older patients with acute myeloid leukemia not considered fit for intensive chemotherapy. Blood 2006; 108: 3262–3270. *13. Weisberg E, Boulton C, Kelly LM et al. Inhibition of mutant FLT3 receptors in leukemia cells by the small molecule tyrosine kinase inhibitor PKC412. Cancer Cell 2002; 1: 433–443. 14. Stone RM, DeAngelo DJ, Klimek V et al. Patients with acute myeloid leukemia and an activating mutation in FLT3 respond to a small-molecule FLT3 tyrosine kinase inhibitor, PKC412. Blood 2005; 105: 54– 60. 15. Stone RM, Fischer T, Paquette R et al. Phase IB study of PKC412, an oral FLT3 kinase inhibitor, in sequential and simultaneous combinations with daunorubicin and cytarabine (DA) induction and high-dose cytarabine consolidation in newly diagnosed adult patients (pts) with acute myeloid leukemia (AML) under age 61. Blood 2006; 108: 50a. [abstract 157]. *16. Levis M, Smith D, Beran M et al. A randomized, open-label study of lestaurtinib (CEP-701), an oral FLT3 inhibitor, administered in sequence with chemotherapy in patients with relapsed AML harboring FLT3 activating mutations: clinical response correlates with successful FLT3 inhibition. Blood 2005; 106: 121a. [abstract 403]. *17. Levis M, Brown P, Smith BD et al. Plasma inhibitory activity (PIA): a pharmacodynamic assay reveals insights into the basis for cytotoxic response to FLT3 inhibitors. Blood 2006; 108: 3477–3483. 18. Nakano Y, Kiyoi H, Miyawaki S et al. Molecular evolution of acute myeloid leukaemia in relapse: unstable N-ras and FLT3 genes compared with p53 gene. British Journal of Haematology 1999; 104: 659–664. *19. Shih LY, Huang CF, Wu JH et al. Internal tandem duplication of FLT3 in relapsed acute myeloid leukemia: a comparative analysis of bone marrow samples from 108 adult patients at diagnosis and relapse. Blood 2002; 100: 2387–2392. 20. Cloos J, Goemans BF, Hess CJ et al. Stability and prognostic influence of FLT3 mutations in paired initial and relapsed AML samples. Leukemia 2006; 20: 1217–1220. *21. Clark JJ, Cools J, Curley DP et al. Variable sensitivity of FLT3 activation loop mutations to the small molecule tyrosine kinase inhibitor MLN518. Blood 2004; 104: 2867–2872. *22. Cools J, Mentens N, Furet P et al. Prediction of resistance to small molecule FLT3 inhibitors: implications for molecularly targeted therapy of acute leukemia. Cancer Research 2004; 64: 6385–6389. 23. Heidel F, Solem FK, Breitenbuecher F et al. Clinical resistance to the kinase inhibitor PKC412 in acute myeloid leukemia by mutation of Asn-676 in the FLT3 tyrosine kinase domain. Blood 2006; 107: 293– 300. 24. Levis M, Pham R, Smith BD et al. In vitro studies of a FLT3 inhibitor combined with chemotherapy: sequence of administration is important to achieve synergistic cytotoxic effects. Blood 2004; 104: 1145–1150.
2 Can FLT3 inhibitors overcome resistance in AML? Winnie F. Tam
PhD
D. Gary Gilliland *
PhD, MD Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA Dana-Farber Cancer Institute, Howard Hughes Medical Institute, Harvard Medical School, Boston, MA, USA
The identification of FLT3 mutations across a range of the cytogenetic subgroups of AML has opened up the possibility of a targeted therapeutic approach with broad applicability. Four agents are currently in clinical trials, at least 3 of which have both sufficient activity against AML and sufficiently acceptable toxicity profiles to support continued efforts to refine their inclusion into therapeutic regimens for AML. Better understanding of the genetics of inherent and acquired resistance is needed to guide development of second-generation agents. Optimizing the integration of FLT3 inhibitor therapy with chemotherapy has the potential both to decrease toxicity and improve response. Key words: FLT3 inhibitors; resistance; staurosporine; midostaurin; PKC412; tandutinib; MLN 518; lestaurtinib; CEP701; SU11248; AML.
INTRODUCTION FLT3 is a class III receptor tyrosine kinase (RTK) expressed on hematopoietic progenitors. It is normally inactive when not bound to its ligand, and is autoinhibited by a juxtamembrane domain that folds structurally into its catalytic site. FLT3 is mutated in about 30% of the patiens with acute myelogenous leukemia (AML); these are lossof-function mutations in the autoinhibitory domain in the form of internal tandem duplications near the juxtamembrane region, or point mutations in the activation loop. Either type of mutation results in constitutive activation of the FLT3 tyrosine kinase in the absence of ligand.1
* Department of Medicine, Brigham and Women’s Hospital, 1 Blackfan Circle, Room 5.0210, Boston, MA 02115, USA. Tel.: þ1 (617) 355 9092; Fax: þ1 (617) 355 9093. E-mail address: [email protected] (D. Gary Gilliland). 1521-6926/$ - see front matter ª 2007 Published by Elsevier Ltd.
14 W. F. Tam and D. Gary Gilliland
FLT3 internal tandem duplications (ITDs) have been identified in virtually every subtype of leukemia including the different cytogenetic risk groups.2 Certain favorable risk groups have a relatively high proportion, including almost 40% in patients with acute promyelocytic leukemia. The mutation is also common in several of the intermediate risk groups, including those with normal cytogenetics. It is not rare in patients that have adverse risk cytogenetics, but importantly, in any of the cytogenetic risk groups, the FLT3 mutation appears to confer a poor prognosis. For example, nearly all poor-risk, ITD-positive patients will relapse within a year, whereas poor-risk ITD-negative patients have a relapse-free survival of about 30% at 1 year. FLT3 positive patients are thus a prognostic group in which molecularly targeted therapy could have a significant therapeutic impact. FLT3 INHIBITORS CURRENTLY IN CLINICAL TRIALS Four different FLT3 inhibitors have reached clinical trials in AML: MLN-518/tandutinib, a quinazolone compound from Millennium;, PKC-412/midostaruin, a benzoyl staurosproine from Novartis, CEP-701/lestaurtinib, an indolocarbazole from Cephalon, and an indolinone Su-11248, initially developed by SuGen, now at Pfizer, which has been associated with cardiotoxic effects3, and has primarily been evaluated in the context of solid tumors.4,5 PKC-412 from Novartis is benzoyl staurosporine.6 The indolocarbazole derivatives from Cephalon were developed in collaboration with Donald Small and colleagues at Hopkins.7 Millennium’s MLN-518 also known as tandutinib, has a 50% inhibitory concentration (IC50) for FLT3, PDGF receptor, and c-KIT of approximately 30 nanomolar (nM), is metabolically stable, and is orally bioavailable. Research groups have shown that tandutinib is efficacious in murine models of FLT3 ITD-induced disease.8 In work on tandutinib, there is in vitro inhibition of FLT3 in cell culture systems with IC50s for inhibition of autophosphorylation of about 30 nM. Preparations from patients with FLT ITDs treated with the drug at doses of 525 mg bid show reduction in phospho-FLT3 content within several hours after initiation of therapy that persists for several days. These data were reported as part of a phase I, open-label trial in 40 patients with relapsed or refractory AML or high risk myelodysplastic syndrome (MDS). In this study, FLT3-ITD mutations were not required as a criterion for enrollment. A single agent, median tolerated dose (MTD) of 525 mg bid was established. The dose-limiting toxicity was reversible; muscle weakness and pharmacodynamics were consistent with an in vivo IC90 of about 350 nM.9 A phase II trial also conducted with this group, using the MTD from the prior study of 525 mg bid in 25 FLT3-ITDpositive patients, showed more than a 50% decrease in bone marrow blasts in 11/25, and 1 patient had a partial CR.9,10 Several patients in this study had dramatic reductions in blast percentage in the peripheral blood, for example from 39,000/mL down to 400/mL or from 12,000/mL down to nearly 0/mL. These are impressive responses in the peripheral blood. The responses in the bone marrow were less dramatic, exhibiting a reduction in blast percentage from 91% to 62% and 80% to 15% on day 28. Other FLT3 inhibitors as well have showed similar responses.3,11,12 DeAngelo and colleagues have followed up these initial results with a phase I/II study of MLN518 plus standard induction chemotherapy in newly diagnosed AML patients. The schema was standard 3 þ 7, daunoribicin and cytosine arabinoside induction with concomitant MLN518 given days 0 to 14 with an option for a second induction if there was persistent leukemia from the bone marrow aspirate and biopsy at day 14. The overall response rate in this study was 70% with 21 of 30 patients achieving a CR.9 The MLN518 was quite well tolerated in combination with the
Can FLT3 inhibitors overcome resistance in AML? 15
standard induction, demonstrating the usual nausea, diarrhea, and vomiting as adverse events. Neutropenia, thrombocytopenia, and a prolonged QTc were the most common grade 3 adverse events and occurred in about 10% of the patients. This suggests that this drug has clinical activity with chemotherapy and that further studies may be warranted to characterize it in randomized trials. PKC412, or midostaurin was developed by Novartis, and is N-benzoylstaurosporine. It was originally developed as a vascular endothelial growth factor (VEGF) and protein kinase C (PKC) inhibitor as the title indicates. A phase I trial was completed, and PKC412 was found to be a FLT3 inhibitor with an IC50 of less than 10 nM. PKC412 also specifically inhibits the growth of leukemic cell lines that are made factor-independent by transfection of the constitutively activating FLT3 mutations, either ITD or D835Y point mutations. It has exhibited activity in FLT3-ITD-induced disease in murine bone marrow transplantation models.1,13 A phase II trial with PKC412 in AML conducted by Richard Stone and colleagues on 25 patients demonstrated greater than 50% reduction in bone marrow blasts in 25% of patients. Two patients had less fewer than 5% blasts, 1 at day 28 and 1 at day 60. Peripheral blood blasts were reduced by more than 70%, including 3 patients who showed a reduction in peripheral blood blast percentage from 110,000/mL to 0, 61,000/mL to 0 and 46,000/mL to 0 at days 29, 57, and 51. The peripheral blood response was again more impressive than the bone marrow response. Seven patients (35%) appeared to have clinical benefit.14 The phase II trial led to a phase IB study of PKC412 in combination with daunorubicin and cytarabine in newly diagnosed patients. The total CR rate was 71% and it was significantly higher in those patients who had FLT-ITD mutations. Eleven of 12 patients went into CR, suggesting that there may be added benefit in patients that are mutant as opposed to wild type. Among patients who responded, 4 subsequently relapsed. These occurred relatively rapidly at 7, 7, 10, and 15 months respectively, but 7 of 11 remain in CR with follow-up of 3 to 15 months.15 It is problematic that the frequent appearance of rapid relapse in responding patients has also emerged in the treatment of chronic myelogenous leukemia (CML) blast crisis with imatinib. The Cancer and Leukemia Group B (CALGB), Southwest Oncology Group (SWOG), and Eastern Cooperative Oncology Group (ECOG) plan to enter into an intergroup study in which the use of PKC412 will be randomized with patients treated with induction chemotherapy with standard 3 þ 7. Patients will be under the age of 60 years and must have mutated FLT3. The study design requires access to the screening of a large number of patients, and the protocol is still in development. CEP701, or lestaurtinib developed by Cephalon, is an indolocarbazole derivative. It has good oral bioavailability. It has a long half-life in murine model systems, and Dr. Small and his colleagues at Johns Hopkins showed in murine model systems that it is a potent inhibitor of FLT3. Reduction in the phospho-FLT3 signal can be seen several hours after initiation of therapy and then recrudescence of the signal at about 12 hours. In a model system using a murine cell line containing the ITD mutation injected into syngeneic recipients, the drug is effective at prolonging survival compared with placebo controls.7,16 Based on these preclinical platforms, a randomized openlabel study was conducted by Dr. Small and colleagues. This study demonstrated that clinical response appears to correlate well with FLT3 inhibition. The trial design took AML patients in first relapse with FLT3 mutations, randomized them to receive either chemotherapy alone or chemotherapy plus CEP701, days 7 through 42. Patients in the control arm were eligible for crossover after assessing initial response. Chemotherapy was based on remission duration. Patients with a remission of 1 to 6 months were treated with mitoxantrone, etoposide and cytarabine. Those with a remission of
16 W. F. Tam and D. Gary Gilliland
6 to 24 months were treated with high dose cytarabine.16 In the 22 patients treated in the chemotherapy plus CEP701 arm, 10 had either full or partial CR (5 and 5), and 9 had no response. Among those 22 patients in the chemotherapy arm, 6 had either full or partial CR (3 and 3) and 15 had no response, suggesting a trend toward efficacy in the chemotherapy þ CEP701 arm. However, in vitro, regardless of mutational status of FLT3, 8 patients who were full or partial responders could also show pharmacodynamically that the target was inhibited in vivo, whereas patients that did not have in vitro sensitivity or in which FLT3 could not be inhibited in vivo were less likely to have responses. This suggests that response correlates with the degree of FLT3 inhibition and that pharmacodynamic monitoring or pretrial assessment may be of value in predicting which patients will respond in future trials. CEP701 also shows cytotoxic activity in a large proportion of AML patients that have FLT3 mutations.17 Sustained plasma levels can be obtained in about 60% of patients with 80 mg bid. The plasma FLT3 inhibitory activity combined with in vitro susceptibility to CEP701 shows a high correlation with clinical response in these trials, suggesting that this may offer benefit to those patients that have FLT3 activating mutations. This is also being tested in a trial that was initiated in Europe. None of the four FLT-3 inhibitors currently in clinical trials offers obvious advantages over the others in terms of response or side effect profile. Most of these appear to be very well tolerated with modest toxicities; using pharmacodynamic estimates, they all appear to inhibit the pharmacologic target. All appear to be active in treating patients with FLT3 mutations, but the most impressive clinical responses have been a reduction of peripheral blood blast percentage which has been more impressive than the reduction in bone marrow blast percentage. Patients that overexpress the wild type also respond, and resistance develops rapidly in those patients who do respond. GUIDING THE FUTURE DEVELOPMENT OF FLT3 INHIBITORS: OVERCOMING RESISTANCE Understanding the background of the activity of FLT3 inhibitors, several topics should guide the next steps of their development. These include undestanding why peripheral blood responses are consistently more impressive than bone marrow responses; the reason why FLT3 inhibitors do not seem to work as effectively as imatinib for CML blast crisis, and the basis for rapid development of resistance. Increasing potency of FLT3 inhibitors may be of value if it can be tolerated without toxicity in patients with AML, and there are several companies that are working actively to develop more potent inhibitors. One potential explanation for the bone marrow versus peripheral blood responses may be microenvironmental resistance to FLT3 inhibitors. Growth factors or stromal cells in the bone marrow may rescue leukemic cells from FLT3 inhibition. If that is so, then in the peripheral blood where cells do not have stromal support, leukemic cells would be highly susceptible to inhibition with FLT3 inhibitors. In the bone marrow, where leukemic cells can be protected by growth factor support, they would be less susceptible to induction of cell death. This model allows consideration of potential solutions, such as trying to mobilize bone marrow blasts in advance of treatment with FLT3 inhibitors, using chemotherapy or growth factors for mobilization, or more elaborate strategies like CXCR4 antagonists or stromal derived factor-1 (SDF1) antagonists that would mobilize blasts into the peripheral blood. The reason why FLT3 inhibitors are not apparently as efficacious in AML as imatinib in the treatment of CML in blast crisis is problematic. In BCR-ABL positive disease, the
Can FLT3 inhibitors overcome resistance in AML? 17
stable phase is associated with expression of BCR-ABL in all cells. The cells that progress to blast crisis may be associated with additional mutations. But nonetheless, in blast crisis, every malignant clonal cell will have BCR-ABL present and these cells are susceptible to inhibition with imatinib, which can induce at least transient CRs until resistance develops. (Figure 1) The FLT3-ITDs mutations in AML are different because, in at least some patients, they may be acquired late in the disease. The status of FLT3ITD in paired diagnosis and relapse samples is an example. Some patients are wild type at diagnosis and at time of relapse; some patients start as wild types and acquire ITD at the time of relapse; some patients have the ITD initially and are wild type at time of relapse; and then of course some patients will have the ITD at diagnosis and at relapse. The ITD can therefore be present at the time of initial therapy but relapse with the clone of cells that lacks the FLT3-ITD2,18–20, differentiating it from BCR-ABL disease. If the FLT3 mutation is present early in disease pathogenesis then the inhibitor could Stable Phase
BCRABL
BCR -ABL BCR -ABL BCR -ABL BCR -ABL
Blast crisis Additional mutations
BCR -ABL BCR -ABL BCR -ABL
imatinib
CR
BCR -ABL
Figure 1. CML in blast crisis can be treated with imatinib because all cells harbor the BCR-ABL mutations. In contrast, FLT3-ITD may be acquired late in disease progression such that not all leukemic blasts have the FLT3-ITD mutations. Thus, treatment with FLT3-ITD inhibitors is not capable of eradicating all leukemia cells resulting in resistance or relapse of disease. CML malignant clones express BCR-ABL in early disease stage and later in blast crisis. For this reason imatinib may be effective in refractory disease because these clones are sensitive to imatinib. FLT3 inhibitors may not be as efficacious in AML as imatinib for treatment of CML blast crisis because the FLT3-ITD mutations can occur at any stage of the disease (see illustration below).
18 W. F. Tam and D. Gary Gilliland
potentially eradicate the leukemic clone leading to CR. However, if FLT3 is acquired as a secondary event in disease pathogenesis, then not all leukemia cells will have the FLT3-ITD. In that situation, even if the inhibitor is effective, the patient is left with a resistant clone that is FLT3-ITD negative and could lead to early relapse. The use of FLT3 inhibitors is unlikely to circumvent this problem, and so combination therapy will be important. In those AML patients who do respond to FLT3 inhibitors, resistance develops rapidly due to increased drug clearance, inherent resistance and the acquisition of resistance mutations analogous to imatinib resistance BCR-ABL mutations. Inherent resistance can be documented with tandutinib in the form of variable sensitivity of the FLT3 activation loop mutations to this particular inhibitor.21 The ITD mutation has an IC50 in this particular cellular context of about 0.550 mM whereas the D835Ys or D835Vs are very highly resistant, with IC50 > 10 mM. These results indicate that it is necessary to think in advance about genotyping patients to predict response to therapy, especially therapy based on FLT3 inhibition. It is also necessary to look at all the activation loop alleles in terms of their sensitivity or spectrum of sensitivity to the inhibitors that are being used in clinical trials. Jahn Cools has examined acquired resistance to FLT3 inhibition using PKC412 resistance mutations and has identified several alleles that confer resistance to PKC412.22 Figure 2 illustrates several of these interacting with the PKC412 molecule where the G697 or N676 mutations can presumably displace PKC from the catalytic pocket. These same two mutations also confer a high degree of resistance to the other three inhibitors discussed above. These potential resistance alleles need to be considered in advance. Thomas Fisher and colleagues, in a clinical trial using PKC412 to treat AML, have identified N676D mutations as conferring clinical resistance.23 Second generation drugs can be proactively developed that are more potent and that will overcome these resistance point mutations if this problem is anticipated. As described above, in vitro analysis shows that there are mutations that confer
Figure 2. Acquired resistance: modeling PKC412 resistance mutations on FLT3 structure. In this model acquired mutations G697 and N676 can displace the inhibitor PKC412 from blocking the activating site, and in this case, the receptor remains constitutively active. The stick figure in green is the PKC412 molecule.22
Can FLT3 inhibitors overcome resistance in AML? 19
a high degree of resistance to PKC412, and 2 of these mutations also confer high resistance to other FLT3 inhibitors, including CEP701 and tandutinib (MLN518). Compounds that will override these resistance alleles should be developed. The next step in developing the use of FLT3 inhibitors to overcome inherent resistance in AML is to use combination therapies. This includes working out the optimal sequencing of chemotherapy and FLT3 inhibitors. For example, Dr. Small and colleagues at Hopkins have developed some in vitro data that suggests that giving chemotherapy first may have some cytotoxic advantage24, though extrapolation into human patients may be problematic. In Dr. Stone’s phase IB trial of PKC412, discussed above15, significant toxicity was encountered when PKC412 was given concurrently with induction chemotherapy. As a result, the study drug is now being sequenced in after the chemotherapy. Along with decreasing toxicity of the combined regimen, this approach may also mobilize the blasts from the microenvironmental protection and improve marrow response. SUMMARY AND CONCLUSIONS The identification of FLT3 mutations across a range of the cytogenetic subgroups of AML has opened up the possibility of a targeted therapeutic approach with broad applicability. However, to guide development of second generation agents, better understanding is needed of the genetics of inherent and acquired resistance, the microenvironmental niche and other factors that appear to shelter blasts in the marrow from FLT3 inhibition in the peripheral blood, and the processes of rapid relapse after initial response. Optimizing the integration of FLT3 inhibitor therapy with chemotherapy has the potential both to decrease toxicity and improve response. REFERENCES 1. Kelly LM, Liu Q, Kutok JL et al. FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myeloproliferative disease in a murine bone marrow transplant model. Blood 2002; 99: 310–318. *2. Kottaridis PD, Gale RE, Frew ME et al. The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy: analysis of 854 patients from the United Kingdom Medical Research Council AML 10 and 12 trials. Blood 2001; 98: 1752–1759. *3. Fiedler W, Serve H, Dohner H et al. A phase 1 study of SU11248 in the treatment of patients with refractory or resistant acute myeloid leukemia (AML) or not amenable to conventional therapy for the disease. Blood 2005; 105: 986–993. 4. Motzer RJ, Rini BI, Bukowski RM et al. Sunitinib in patients with metastatic renal cell carcinoma. The Journal of the American Medical Association 2006; 295: 2516–2524. 5. Motzer RJ, Hoosen S, Bello CL et al. Sunitinib malate for the treatment of solid tumours: a review of current clinical data. Expert Opinion on Investigational Drugs 2006; 15: 553–561. 6. Fabbro D, Ruetz S, Bodis S et al. PKC412–a protein kinase inhibitor with a broad therapeutic potential. Anti-Cancer Drug Design 2000; 15: 17–28. *7. Levis M, Allebach J, Tse KF et al. A FLT3-targeted tyrosine kinase inhibitor is cytotoxic to leukemia cells in vitro and in vivo. Blood 2002; 99: 3885–3891. *8. Kelly LM, Yu JC, Boulton CL et al. CT53518, a novel selective FLT3 antagonist for the treatment of acute myelogenous leukemia (AML). Cancer Cell 2002; 1: 421–432. 9. DeAngelo DJ, Amrein PC, Kovacsocics TJ et al. Phase 1/2 study of tandutinib (MLN518) plus standard induction chemotherapy in newly diagnosed acute myelogenous leukemia (AML). Blood 2006; 108: 51a. [abstract 158].
20 W. F. Tam and D. Gary Gilliland 10. De Angelo DJ, Stone R, Heaney ML et al. Phase II ealuation of the tyrosine kinase inhibitor MLN518 in patients with acute myeloid leukemia (AML) bearing a FLT3 internal tandem duplication (ITD) mutation. Blood 2004; 104: 496a. [abstract 1792]. 11. Stone RM, De AJ, Galinsky I et al. PKC 412 FLT3 inhibitor therapy in AML: results of a phase II trial. Annals of Hematology 2004; 83(Suppl. 1): S89–S90. 12. Knapper S, Burnett AK, Littlewood Tet al. A phase 2 trial of the FLT3 inhibitor lestaurtinib (CEP701) as first-line treatment for older patients with acute myeloid leukemia not considered fit for intensive chemotherapy. Blood 2006; 108: 3262–3270. *13. Weisberg E, Boulton C, Kelly LM et al. Inhibition of mutant FLT3 receptors in leukemia cells by the small molecule tyrosine kinase inhibitor PKC412. Cancer Cell 2002; 1: 433–443. 14. Stone RM, DeAngelo DJ, Klimek V et al. Patients with acute myeloid leukemia and an activating mutation in FLT3 respond to a small-molecule FLT3 tyrosine kinase inhibitor, PKC412. Blood 2005; 105: 54– 60. 15. Stone RM, Fischer T, Paquette R et al. Phase IB study of PKC412, an oral FLT3 kinase inhibitor, in sequential and simultaneous combinations with daunorubicin and cytarabine (DA) induction and high-dose cytarabine consolidation in newly diagnosed adult patients (pts) with acute myeloid leukemia (AML) under age 61. Blood 2006; 108: 50a. [abstract 157]. *16. Levis M, Smith D, Beran M et al. A randomized, open-label study of lestaurtinib (CEP-701), an oral FLT3 inhibitor, administered in sequence with chemotherapy in patients with relapsed AML harboring FLT3 activating mutations: clinical response correlates with successful FLT3 inhibition. Blood 2005; 106: 121a. [abstract 403]. *17. Levis M, Brown P, Smith BD et al. Plasma inhibitory activity (PIA): a pharmacodynamic assay reveals insights into the basis for cytotoxic response to FLT3 inhibitors. Blood 2006; 108: 3477–3483. 18. Nakano Y, Kiyoi H, Miyawaki S et al. Molecular evolution of acute myeloid leukaemia in relapse: unstable N-ras and FLT3 genes compared with p53 gene. British Journal of Haematology 1999; 104: 659–664. *19. Shih LY, Huang CF, Wu JH et al. Internal tandem duplication of FLT3 in relapsed acute myeloid leukemia: a comparative analysis of bone marrow samples from 108 adult patients at diagnosis and relapse. Blood 2002; 100: 2387–2392. 20. Cloos J, Goemans BF, Hess CJ et al. Stability and prognostic influence of FLT3 mutations in paired initial and relapsed AML samples. Leukemia 2006; 20: 1217–1220. *21. Clark JJ, Cools J, Curley DP et al. Variable sensitivity of FLT3 activation loop mutations to the small molecule tyrosine kinase inhibitor MLN518. Blood 2004; 104: 2867–2872. *22. Cools J, Mentens N, Furet P et al. Prediction of resistance to small molecule FLT3 inhibitors: implications for molecularly targeted therapy of acute leukemia. Cancer Research 2004; 64: 6385–6389. 23. Heidel F, Solem FK, Breitenbuecher F et al. Clinical resistance to the kinase inhibitor PKC412 in acute myeloid leukemia by mutation of Asn-676 in the FLT3 tyrosine kinase domain. Blood 2006; 107: 293– 300. 24. Levis M, Pham R, Smith BD et al. In vitro studies of a FLT3 inhibitor combined with chemotherapy: sequence of administration is important to achieve synergistic cytotoxic effects. Blood 2004; 104: 1145–1150.