FLT3: AT arget for Molecular Therapy in AML by pLdevf2h

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									FLT3: A Target for Molecular
     Therapy in AML
          Bill Blum, MD
       Division of Hem/Onc
      Siteman Cancer Center
           FLT 3 Background
• “FMS-like tyrosine kinase”

• Member of Class III receptor tyrosine kinases (RTK) that
  includes
   – FMS, c-kit, PDGFR

• Five Ig-like domains in extracell regions

• Ligand binding induces dimerization

• Activated RTKs phosphorylate signaling molecules
  leading to downstream activation of STAT-5, MAPK, akt
                       FLT 3
                                                 Chromosome 13
• Region on Chromosome 13q12 in humans

• Activating mutations have been noted in
  patients with AML
   – mostly internal tandem duplications (ITD)
   – less common, point mutations in the
     activation loop

• Mutations cluster in the juxtamembrane
  domain of the FLT3 protein
                      FLT 3
• ITD in the juxtamembrane domain of FLT3 have
  been found in myeloid leukemias and
  myelodysplastic syndromes

• Duplicated sequences vary in position in length,
  but are in-frame and result in an elongated product

• ITD are activating mutations

• Association with high WBC, high blast counts
   – and poor prognosis?
               FLT 3-Mutations
               Should we care?
• Conflicting data on prognostic significance of FLT3 ITD in
  AML, but most studies demonstrate reduced DFS for mutants

• FLT3 ITD appear to be more common in patients with
  “intermediate” cytogenetics--and may provide a useful
  measure for detection of minimal residual disease in this
  population (but maybe not) (Blood 2002)

• Increasing age does not appear to be associated with increased
  FLT3 ITD expression, but older patients tolerate induction less
  well and may especially benefit if new therapies to improve
  disease control could be targeted at FLT3 mutants
Gilliland and Griffin, Blood 2002
  FLT 3-Activating Mutations
          in AML
• 20-25% of all AML patients (70%-100% of AML will
  express wild-type FLT3)

• 28-38% of AML patients with normal cytogenetics

• ?higher numbers 35-41% in APL

• higher in de novo (26%) vs. secondary (9%)

• t(8,21)-11%, inv16-2%, MLL-7%, complex-2%
Schnittger, Blood 2002
Thiede, Blood 2002
Thiede, Blood 2002
Thiede, Blood 2002
Thiede, Blood 2002
CALGB data
Whitman, et al
Cancer Research, 2002
82 adults <60yrs, AML,
normal cytogenetics
ITD in 23%
Mutants had inferior DFS,
but OS was not different
8/23 mutants also lacked a
wt allele, ITD/-, and both
DFS and OS were worse
for this subgroup
Whitman, et al, Cancer Research, 2002
Whitman, et al, Cancer Research, 2002
Whitman, et al, Cancer Research, 2002




                   Activation of both physiologic and aberrant signaling
         FLT3- Mouse Models
• FLT3 ITD mutant mice do not develop acute leukemia
   – However, after a latency period of 40-60 days, BALB/c
     mice develop a myeloproliferative syndrome with
     leukocytosis and splenomegaly.
   – Conversely, B6 x C3H (F1) develop T-cell LL with
     median latency of 100 days

• Suggests that FLT3 ITD may not be sufficient to cause
  disease alone and that it may be one of many mutations
  that together can cause leukemia

• Also suggesting that FLT3 inhibitors may not have the
  same dramatic effect as STI571 as single agent therapy
                    FLT3
• Hypothesis:

  – FLT3 ITD is not sufficient to cause AML
    alone, but perhaps it may cooperate with
    additional mutations to cause disease.

  – Likewise FLT3 TK inhibitors are unlikely to
    have as dramatic an effect as STI571 given the
    likelihood that additional mutations would also
    be present
   What additional mutations will lead to AML if
              FLT3 ITD is present?

• Model system of PML-RARA
  – PML-RARA is also not sufficient to cause APL in large
    numbers of mice (Blood 97)
  – Transgenics with PML-RARA (under the control of the
    cathepsin G promoter) first develop a myeloprolif dz,
    but then 15-20% get APL after 6-13 months (Blood 2002)

• The mice that get APL often are noted to have additional
  cytogenetic changes prior to dz progression (PNAS 1997)

• High numbers of APL patients have been reported to have
  FLT3 ITD, so
              FLT3- Mouse Models
• Hypothesis:
  FLT3 ITD may cooperate with PML-RARA
  to cause APL in mice with a short latency time
• Experiment:        Control
                     Arm      Plus
 Transgenic Mice             EGFP         Transplanted
                                        into irradiated
                                        recipients
   PML-                                 Cohorts
   RARA+                                followed for
                              Plus      development of
                                        APL
                           FLT3 ITD
             FLT3 ITD + PML-RARA
• The APL caused by this cooperation is similar to that found
  in murine models of PML-RARA alone, except with
   – 100% penetrance (vs. 20%)
   – short latency period of 7-23 weeks (vs. 6-13 months)


• The diseases in both are transplantable to secondary
  recipients. Both respond to ATRA differentiation (see next slide)

• Conclusion:
   – FLT3 ITD can functionally substitute for the additional mutations
     required for PML-RARA+ mice to develop APL.
   – This suggests practicality in studying new drugs that would abrogate
     all or part of this “cooperation” and thus be effective against AML.
PML-RARA without ATRA   PML-RARA with ATRA




FLT3 ITD + PML-RARA     FLT3 ITD + PML-RARA
without ATRA            with ATRA
Treatment of FLT3 ITD+ AML
• Given the increased relapse rate (vs. wild-type),
  FLT3 mutants may be more chemo-resistant. Can
  this be overcome with high-dose therapy or GVL
  effects?

• Can the kinase itself be specifically targeted and
  thus block downstream signaling?

• Will disruption of related cellular processes also
  serve to disrupt FLT3 signaling?
Given the increased relapse rate (vs. wild-type), FLT3
mutants may be more chemo-resistant. Can this be
overcome with high-dose therapy or GVL effects?

Kottaridis #273                                CR
• 1162 patients (<60)
• 306 (26%) with FLT3 ITD
                                 Chemo
• No diff in CR rate (83/84%)
                                 N= 739                  Allo
                                                         N=98
              3yr DFS       OS
ITD+          46%           58               Auto
                                             N=100
ITD neg       57            66
ITD+          20            28
ITD neg       59            60
Can the kinase itself be specifically targeted and
thus block downstream signaling?

 • New compounds with early data presented
   at ASH 2002
    –   SU 11248
    –   CEP 701
    –   CEP 5214
    –   SU 5614
    –   MLN 518
    –   PKC412
    [1305] A "First in Man" Study of the Safety and PK/PD of
    an Oral FLT3 Inhibitor (MLN518) in Patients with AML or
    High Risk Myelodysplasia.
    Michael C. Heinrich
                                       Preclinical

•   MLN518 = CT53518
•   also inhibits other Class III RTK
•   inhibits both wt and mutant FLT3 at IC50 of 200nM
•   blocks FLT3 ITD dependent signaling via MAP
    kinase, PI3 kinase pathway, induced apoptosis
• leukemia cell lines, murine BMT models
 [1305] A "First in Man" Study of the Safety and PK/PD of
 an Oral FLT3 Inhibitor (MLN518) in Patients with AML
 or High Risk Myelodysplasia.
 Michael C. Heinrich
                                    Phase I


• 28 day course of oral MLN518
   – did not have to have FLT3 ITD+ to enroll
• Initial dose 50mg q12
• Well tolerated
• 2/3 evaluable patients had >50% reduction in blasts
[118] The Protein Tyrosine Kinase Inhibitor SU5614
Inhibits FLT3 and Induces Growth Arrest and Apoptosis in
AML Cells Expressing a Constitutively Active FLT3.
Karsten Spiekermann

• In leukemia cell lines, SU5614 induced growth
  arrest, apoptosis, cell cycle arrest
   – effect seen only in mutant FLT3 activated cells
   – wt cells were not affected


• SU5614 downregulated the activity of the
  hyperphosphorylated FLT3 receptor and its
  downstream targets STAT3, MAPK, STAT5 and
  STAT5 target genes Bcl-X and p21.
[2196] An Innovative Single Dose Clinical Study Shows
Potent Inhibition of FLT3 Phosphorylation by SU11248 In
Vivo: A Clinical & Pharmacodynamic Study in AML pts.
James Foran

• SU11248 targets FLT3, VEGFR2, PDGFR, KIT
• Preclinical
   – inhibits phosphorylation of wt FLT3, mutant
     FLT3 and induces apoptosis in vitro
   – in vivo SQ tumor xenografts and BMT models
     showed dose dependent efficacy and PK/PD
     relationship for inhibition of phosphorylation
     was established
[2196] An Innovative Single Dose Clinical Study Shows
Potent Inhibition of FLT3 Phosphorylation by SU11248 In
Vivo: A Clinical & Pharmacodynamic Study in AML pts.
James Foran
• Phase I, 29 patients enrolled at 5 sites for single-dose trial

• Primary endpoint: >50% inhibition of FLT3
  phosphorylation in 3/6 patients (each dose level). This was
  reached at each dose level above 200mg, including wt and
  mutant pts

• FLT3 modulation was achieved at lower conc for mutants
  than wt (16-139 vs. 60-201ng/ml)(n=5 vs. 8)

• “Reductions” in blast counts also noted
[2195] A Phase I Study of Repeated Oral Dosing with
SU11248 for the Treatment of Patients with Acute Myeloid
Leukemia Who Have Failed, or Are Not Eligible for,
Conventional Chemotherapy.
James Foran


• Phase I, 4 dose levels (oral), 32 patients

• genotyping ongoing at publication
  – 16 wt, 4 mutant patients

• 13/16 patients with detectable PB blasts at onset
  had >50% reductions
[314] Single Agent CEP-701, a Novel FLT-3 Inhibitor,
Shows Initial Response in Patients with Refractory Acute
Myeloid Leukemia.
B. Douglas Smith
• CEP-701 inhibits autophosphorylation of wt and
  constitutively activated FLT3 at IC50 2-3nM
   – results in cell death in AML lines
   – prolongs survival in mouse model
• Phase II
• 12 patients, all failed salvage chemo, 8 were primary
  refractory
• NR in 1st 3 patients, so dose increased
• At higher dose, 4/9 had response
   – WBC 1.2 (80% blasts) d15 WBC 5.0, no blasts
Will disruption of related cellular processes also serve to disrupt
constitutively activated FLT3 signaling?
                                                       HSP client
                                                       proteins
                                                       glucocort-R
   HSP 90                                              progest-R
                                                       estrogen-R
                                                       androgen-R
                                                       retinoid-R
                                                       v-src
                                                       akt
                                                       telomerase
                                                       mutant p53
                                                       her-2/neu
                                                       bcr-abl
                                                       FLT3
from www.conformacorp.com
Cellular pathways potentially
affected by chaperone inhibition




                           from www.conformacorp.com
Minami, Leukemia 2002


                        Inhibitors of HSP 90
                        currently include:
                        Geldanamycin,
                        17AAG,
                        Herbimycin A,
                        Radicicol
                        HA and Radicicol
                        are shown here to
                        preferentially kill
                        FLT3 mutant cells
                        over wild type cells.
                        Both are toxic
                        compounds too
                        unstable for human
                        use.
                Conclusions
• Mutations that activate FLT3 in AML are among
  the most common known to be associated with the
  disease

• These mutations confer adverse risk to patients
  with AML and are commonly found in those with
  normal cytogenetics

• FLT3 mutations are not sufficient to cause AML
  alone, but rather they cooperate with other events
  to cause disease
                 Conclusions
• Selective inhibition of the kinase can be achieved by
  physiologically attainable drug levels with limited
  toxicity

• Early clinical results with these selective inhibitors
  are promising but also suggest that they may be more
  effective when used in combination with other agents

• Pre-clinical data shows that inhibition of FLT3
  mutants can also be achieved by inhibiting the
  chaperone HSP90
                 Conclusions
• Perhaps combinations of multiple “targeted”
  therapies will lead to better disease control at
  reduced toxicity compared to conventional cytotoxics




                   Sohal, et al, Blood prepublished online, Jan 16, 2003

								
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