FLT3: A Target for Molecular Therapy in AML

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FLT3 A Target for Molecular Therapy in AML

• Bill Blum, MD
• Division of Hem/Onc
• Siteman Cancer Center

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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

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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

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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?

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FLT 3-MutationsShould 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

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Gilliland and Griffin, Blood 2002
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FLT 3-Activating Mutationsin 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

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Schnittger, Blood 2002
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Thiede, Blood 2002
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Thiede, Blood 2002

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Thiede, Blood 2002
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Thiede, Blood 2002
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CALGB data Whitman, et al Cancer
Research, 2002 82 adults lt60yrs, 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
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Whitman, et al, Cancer Research, 2002
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Whitman, et al, Cancer Research, 2002
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Whitman, et al, Cancer Research, 2002
Activation of both physiologic and aberrant
signaling
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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

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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

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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

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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 EGFP
Transgenic Mice
Transplanted into irradiated
recipients Cohorts followed for development of APL
PML-RARA
Plus FLT3 ITD
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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.

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PML-RARA without ATRA
PML-RARA with ATRA
FLT3 ITD PML-RARA without ATRA
FLT3 ITD PML-RARA with ATRA
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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?

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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
• 1162 patients (lt60)
• 306 (26) with FLT3 ITD
• No diff in CR rate (83/84)

CR
Chemo N 739
Allo N98
3yr DFS OS ITD 46 58 ITD
neg 57 66 ITD 20 28 ITD neg 59 60
Auto N100
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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

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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.Mic
hael 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

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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.Mic
hael 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 gt50 reduction in
  blasts

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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.

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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

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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 gt50 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)(n5 vs.
  8)
• Reductions in blast counts also noted

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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 gt50 reductions

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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

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Will disruption of related cellular processes
also serve to disrupt constitutively activated
FLT3 signaling?
HSP client proteins glucocort-R progest-R estrogen
-R androgen-R retinoid-R v-src akt telomerase muta
nt p53 her-2/neu bcr-abl FLT3
HSP 90
from www.conformacorp.com
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Cellular pathways potentially affected by
chaperone inhibition
from www.conformacorp.com
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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.
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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

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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

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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