LBH589: highly effective in rituximab-resistant lymphomas and enhancement of anti-tumor activity of bortezomib, other chemotherapy agents, and anti-CD20 monoclonal antibodies

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LBH589highly effective in rituximab-resistant
lymphomas and enhancement of anti-tumor activity
of bortezomib, other chemotherapy agents, and
anti-CD20 monoclonal antibodies

• Mohammad Muhsin Chisti
• Mentor Francisco Hernandez- Illizaliturri
• Other Authors Ilir Maraj and Myron Czuczman
• University at Buffalo Sisters of Charity
  Hospital
• Roswell Park Cancer institute, Buffalo, NY

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Introduction

• Deacetylases are enzymes that remove the acetyl
  groups from target proteins (histones and
  non-histones ) leading to regulation of gene
  transcription and other cellular processes.
• LBH589 is a novel and potent DAC class I and II
  inhibitor undergoing pre-clinical and clinical
  testing.

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There are 2 Classes of DACs ( I and II ), Which
Act on Different Target Proteins
There are 2 main classes of DACs
Class I DACs act on HISTONES and TRANSCRIPTION FAC
TORS located in the nucleus
Class II DACs act on NON-HISTONE proteins
located in the cytoplasm (e.g. HDAC6)
HDAC1
HDAC2
HDAC3
HDAC6
HDAC8
HDAC4
HDAC5
HDAC7
HDAC7
HDAC9
HDAC10
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Genetic Variations and Epigenetic Changes Can
Both Contribute to Oncogenesis
GENETIC
DNA
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Acetylation of Histones by HAT Allows Gene
Expression
Acetylation by histone acetyltransferases (HATs)
allows transcription and gene expression
HAT
Transcription factors
HISTONE ACETYLATION
Acetylated Histone Open chromatin Transcription
factors can access DNA
Deacetylated Histone Closed chromatin
Transcription factors cannot access DNA
Ac acetyl group
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Deacetylation of Histones by HDAC Can Prevent
Gene Expression
Acetylation by histone acetyltransferases (HATs)
allows transcription and gene expression
HAT
Transcription factors
HISTONE ACETYLATION
Acetylated Histone Open chromatin Transcription
factors can access DNA
Deacetylated Histone Closed chromatin
Transcription factors cannot access DNA
HISTONE DEACETYLATION
HDAC
Deacetylation by histone deacetylases (HDACs) can
prevent transcription and gene expression
Ac acetyl group HDAC depicts a class I
deacetylase
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In Normal Cells, Balanced HAT and HDAC Activity
Results in Regulated Gene Expression
Histone deacetylation prevents gene expression
Histone acetylation allows gene expression
HDAC
HAT
TF
Deacetylation
Acetylation
Normal Cell
Ac acetyl group TF transcription factors HDAC
depicts a class I deacetylase
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In Tumor Cells, Imbalanced HAT and HDAC Activity
Can Result in Deregulated Gene Expression
IncreasedHDAC Activity
Decreased HAT Activity
HAT
HDAC
HDAC
TF
HDAC
Decreased Tumor Suppressor Gene Activity (p21,
p27)
TumorCell
Ac acetyl group TF transcription factors HDAC
depicts a class I deacetylase
Unchecked CellGrowth and Survival
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HDAC Inhibition Restores Gene Expression in Tumor
Cells
DAC Inhibition Increases Acetylation of Histones
HAT
DAC Inhibitor
TF
Increased Tumor Suppressor Gene Activity (p21,
p27)
Normalized Cell
Ac acetyl group TF transcription factors HDAC
depicts a class I deacetylase
Cell-Cycle Arrest and Differentiation
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DAC Inhibition Induces Cell Death in Tumor Cells,
But Not Normal Cells
DAC Inhibitor
Normal Cell
Tumor Cell
Reversible G2/M Arrest
Loss of cell-cycle control
Apoptosis
No apoptosis
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Deacetylase (DAC) Activity on Proteins is
Associated with Downstream Effects that Promote
Oncogenesis
DACs
DACs
DACs
DACs
DACs
Proteins modulated by DACs
Histone
HIF-1a
?-tubulin
HSP90
p53
DAC depicts individual deacetylases, e.g. HDAC1,
HDAC4, HDAC6
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Pan-DAC Inhibition Interferes with the Multiple
Hallmarks of Cancer
DAC Inhibitor
Proteins modulated by DACs
DAC
DAC
DAC
DAC
DAC
Histone
HIF-1a
?-tubulin
HSP90
p53
DAC depicts individual deacetylases, e.g. HDAC1,
HDAC4, HDAC6
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Pan-DAC Inhibition May Have Potential in Several
Cancers
50 of Cancers
Hematologic Solid Tumors
DAC Inhibitor
Histone
p53
DACs
HSP90
?-tubulin
HIF-1a
CML, Breast, Prostate, NSCLC
Breast, Multiple Myeloma
RCC, Melanoma
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Rationale of combining LBH589 with other agents

• To characterize the role of DAC inhibitors we
  studied the effect of LBH589 when used with
  chemotherapy agents and anti-CD20 monoclonal
  antibodies against a panel of
• RSCL and RRCL
• lymphoma cells isolated from patients

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Methods and Materials

• Cell Lines Used
• Rituximab-sensitive cell lines (RSCL)
• Raji,
• SU-DHL4,
• RL cells
• Rituximab-resistant cell lines (RRCL)
• Raji-2R,
• Raji-4RH,
• RL-4RH
• SU-DHL-4RH.
• Therapy-resistant cell lines were created as
  described in Czuczman et al. (Clin Cancer Res.
  2008 14(5)1561-70).

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Development of rituximab-chemotherapy resistant
models
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• Chemotherapeutic agents used
•  Cisplatin, doxorubicin, vincristine
• Monoclonal Antibodies and other agents Used
• Bortezomib , Rituximab , Veltuzumab (or isotype
  control), Trastuzumab, GX15-070 (Obataclox)

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Exp 1 Viability assays of LBH589 alone or in
combination with chemotherapy or monoclonal
antibodies

• LBH589 concentration used in this experiment was
  0 to 50 ?M.
• 96 well plates were used and NHL cells were
  plated in each well.
• We studied if the sequence of administration
  between LBH589 and mAb or chemotherapy agents
  plays a role in the global anti-tumor activity.
• In dose-sequence studies the treatment with
  LBH589 preceded or followed in vitro exposure to
  the chemotherapy agent or the monoclonal antibody
  by 24 hrs.

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• NHL cell lines were exposed to LBH589 (0 to 50?M)
  only or with CDDP (0 to 10?M), Vincristine (0 to
  10 nM), Doxorubicin (0 to 400nM), boretzomib (1
  to 10nM) rituximab (10?g/ml), veltuzumab
  (10?g/ml) or isotype (10?g/ml) .
• Subsequently NHL cells were treated with mAbs
  (Rituximab, A20, ETR1 or Isotype control),
  chemotherapy drugs (CDDP, Doxorubicin,
  Vincristine, Bortezomib, GX15-070) or RPMI-10
  (Placebo) alone or in combination with LBH589 or
  placebo.

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• Experiment were performed in triplicates.
• After incubation of 48hrs from the initial plate
  set up and 24 hrs after adding the 2nd drug,
  alamar blue was added to the plate (20ml/well).
• Following a 24 and a 48 hour-period of drug
  exposure, changes in mitochondrial potential and
  cell proliferation were determined by alamar blue
  reduction using a kinetic assay measuring
  activity at 15 minute intervals for 24 and 48
  hrs.

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LBH589 induces cell death by both Capase
dependent and independent pathways

• Both RSCL or RRCL are sensitive to LBH589 as
  determined by CellTiter-Glo luminescent
  viability assay.
• The addition of caspase inhibitors Q-VD-Oph or
  ZVAD partially decreased the anti-tumor activity
  of LBH589 only in RSCL.
• In RRCL, known to have low levels of Bax/Bak, the
  primary mechanisms of cell killing appears to be
  caspase independent .

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LBH589 induce cell death in rituximab-chemotherapy
sensitive and resistant B-cell lymphoma cell
lines
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LBH589 enhances the anti-tumor activity of
various chemotherapy agents

• Incubation of RSCL or RRCL with LBH589 and
  cisplatin ,bortezomib and doxorubicin resulted in
  a synergistic decrease in the mitochondrial
  potential at 48 hrs when compared to each agent
  alone.
• RSCL or RRCL cells were plated in 384 well plates
  at a cell density of 0.5x105 cells/ml and exposed
  to LBH589 (0, 0.2, 2 or 20nM) with or without
  CDDP (1, 10 and 100µM), bortezomib (0.1, 1 or
  10nM), or doxorubicin (0.16, 1.6 or 16µM).

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LBH589 enhances the anti-tumor activity of
various chemotherapy agents in rituximab
sensitive and resistant B-cell lymphoma cell
lines
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Exp 2 51Cr release assays (CMC and ADCC)

• Rituximab-resistant cells (RRCL) derived from
  Raji and RL cells are insensitive to
  rituximab-induced CMC as determined by 51Cr
  release assays.
• 51Cr-labeled NHL cells were exposed to rituximab
  or isotype control (10?g/ml) and human serum,
  respectively.
• 51Cr-release was measured and the percentage of
  lysis was calculated.

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• Pre-incubation of a panel of NHL cell lines with
  LBH589 (2 or 20nM) for up to 48 hrs does appear
  to diminish rituximab or veltuzumab (A20) CMC.
• Flow cytometry analysis demonstrate that in vitro
  exposure of RSCL and RRCL resulted in a decrease
  in surface CD20 expression.

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LBH589 appears to diminish the capacity of
anti-CD20 mAbs to induce complement mediated
lysis (CMC)
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Exp 3Viability assays evaluating LBH589 in
combination with proteasome inhibitors or mab

• Malignant B-cells were isolated by MACS sorting
  (negative selection) from pre-treatment biopsy
  tissue obtained from B-cell lymphoma patients
• Tissue was mechanically disrupted and cells
  passed through a 100 micron cell strainer.
• Lymphocytes were enriched by density
  centrifugation.
• B-cells were isolated from enriched lymphocytes
  by MACS separation using a human B-cell Isolation
  Kit II (Miltenyi Biotec).
• B-cell purity was assessed by flow cytometry
  using antibodies to CD19 and CD20.
• Greater than 95 pure CD19 cells were obtained
  from each biopsy processed.

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• Tumor derived lymphoma cells isolated from biopsy
  specimens were exposed ex vivo to LBH589 with or
  without bortezomib or mab and incubated for 24 or
  48 hrs.
• Cellular viability following exposure to LBH589
  (2 to 20nM) and or Bortezomib or mab (1 to 10nM)
  was determined using CellTiter-Glo luminescent
  viability assay (Promega).
• Following background subtraction, ATP content
  (viability) was determined as a percentage of the
  luminescence produced by control, vehicle treated
  cells.

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Dose-sequence effects of LBH589 on chemotherapy
agents
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Dose-sequence effects of LBH589 on monoclonal
antibodies activity
Pre-incubation of NHL cells with LBH589 prior to
bortezomib or mAb exposure leads to optimal
killing by each agent compared to other sequences
of administration
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Ex-vivo effects of LBH589 /- Bortezomib in
tumor cells derived from lymphoma patients
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• Malignant B-cells purified from biopsies from
  patients with follicular cell lymphoma or
  Hodgkins lymphoma displayed a variable degree of
  sensitivity to LBH589 that was independent of
  their sensitivity to standard chemotherapeutic
  agents and/or rituximab.
• In addition, the addition of LBH589 to Bortezomib
  resulted in significant synergistic activity when
  compared to either agent alone.
• Combination of LBH589 and bortezomib resulted in
  significant anti-tumor activity in follicular,
  Hodgkin and diffuse large B-cell lymphoma.

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Exp 4 Reverse Transcription (RT) polymerase
chain reaction (PCR)

• NHL cells were exposed to LBH589 (20nM) or DMSO
  control for 24 or 48 hrs.
• Bax, Bak, Bcl-2, Mcl-1, Bcl-XL, Bik were
  specifically amplified from cDNAs created by
  reverse transcription from RNA extracted from 0.5
  2 x 106 cells in log phase growth.
• PCR products were electrophoresed on 1.2 agarose
  gels containing ethidium bromide and visualized
  by UV transillumination.

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LBH589 down-regulates Bcl-Xl expression in NHL
cells
RL
RL4RH
In vitro exposure of rituximab-chemotherapy
sensitive (RSCL) or resistant (RRCL) cell lines
modifies the genetic expression of BCL-2 family
members, primarily Bcl-XL and MCL
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Result

• LBH589 was active as a single agent against RSCL,
  RRCL or patient-derived primary tumor cells.
• In addition, Bcl-XL gene down-regulation was
  observed following exposure to LBH859.
• On the other hand, upregulation of Bak and
  downregulation of Mcl-1 were observed following
  proteasome inhibition.

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Conclusions

• LBH589 appears to mediate cell death by both
  caspase-dependent and independent mechanisms.
• LBH589 is capable of killing therapy-sensitive
  and therapy-resistant aggressive B-NHL cells,
  including those derived from patients with
  various subtypes of lymphoma
• In vitro, LBH589 appears to potentiate the
  anti-tumor activity of bortezomib, cisplatin, and
  rituximab against RRCL or RSCL and in tumor cells
  derived from patients with B-cell lymphoma
  (bortezomib only)

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• It appears to disrupt the balance between
  pro-apoptotic and anti-apoptotic potential
• Our findings strongly suggest that LBH589 added
  to anti-CD20 and/or chemotherapy results in a
  novel and potent treatment strategy against
  B-cell lymphoma.
• Blood (ASH Annual Meeting Abstracts), Nov 2008
  112 4984. 
• Journal of Clinical Oncology J Clin Oncol 2715s,
  2009 (suppl abstr 8576) (ASCO Annual Meeting ) 
  Abstract No 8576
• Blood (ASH Annual Meeting Abstracts) 2009 114
  Abstract 2715
• New York ACP 2010, poster session, Mount Sinai.

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• Research, in part, supported as part of a
  subproject on NIH PO1 grant CA103985-1 awarded to
  the Garden State Cancer Center, Belleville, NJ
  and NHI R-01 grant R01 CA136907-01A1 awarded to
  Roswell Park Cancer Institute

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Acknowledgement
Dr Francisco Hernandez-Illizaliturri
Dr Myron Czuczman
Dr Michael Krabak
Dr Khalid J Qazi