Objective
Single agent therapies used for the treatment of T1D to date have failed to reverse established
disease in both preclinical animal and human studies. It has become increasingly clear that
reversal strategies for diabetes must both 1) replace cell mass and 2) suppress autoreactive T cell
responses to protect new insulin producing cells from recurrent autoimmunity, as a failure to
correct both conditions ultimately will have little lasting benefit on the disease course. The
research we propose is designed to investigate whether combinatorial use of the Focal Adhesion
Kinase (FAK) inhibitor FAKi and glycolysis inhibitor PFK15 has the ability to reverse established
T1D. Our recent manuscript and preliminary data demonstrate a unique ability to regenerate insulin-
secreting like cells endogenously from pancreatic acinar cells (FAKi) and induce terminal
exhaustion of - cell reactive T cells (PFK15) when used as single agents in models of STZ- induced
diabetes and in prevention studies. However, we believe combinatorial use of these 2 agents will
have the ability to reverse overt diabetes in affected T1D patients, which will be explored in
preclinical mouse studies as described within our proposal. We hypothesize that combinatorial use
of FAKi and PFK15 can reverse T1D onset in vivo by regenerating insulin- secreting like cells
endogenously from acinar cells and protecting new insulin- secreting cells from autoimmune
targeting by inducing exhaustion of - cell reactive T cells. To test this hypothesis, we propose
the following specific aims:
Aim 1: To establish whether FAKi induces trans-differentiation of acinar cells into insulin
secreting - like cells under autoimmune conditions.
Aim 2: To determine whether FAKi in combination with PFK15 can reverse T1D onset in NOD mice.
Background Rationale
It has become increasingly clear that reversal strategies for T1D must both 1) replace cell mass
and 2) suppress autoreactive T cell responses to protect new insulin producing cells from recurrent
autoimmunity. Failure to correct both conditions will result in an inability to reverse disease
effectively, and demonstrates an urgent need to investigate combinatorial approaches to improve
current clinical outcomes. We have identified two novel therapeutic targets that have proven
beneficial as single agents in improving blood glucose (BG) levels in streptozotocin (STZ)- induced
diabetes mouse and non-human primate models (FAKi) and in preventing adoptive transfer (AT) of
diabetes in a model of T1D (PFK15), however their use in combination has not been tested, and is
the premise of our proposal. To replace lost cell mass after onset of T1D, we will convert
pancreatic acinar cells into insulin- secreting like cells by inhibiting the kinase activity of
Focal adhesion kinase (FAK). FAK is a cytoplasmic non-receptor tyrosine kinase involved in
mediating integrin signaling. Our preliminary data provide evidence that treatment of adult mice
with a compound that specifically inhibits kinase activity of FAK results in a remarkable
transdifferentiation of acinar cells into insulin-producing 1 like cells in mice. This is a
significant and relevant theme as acinar cells are the most abundant cell type in the pancreas
(~85%) and thus can serve as a good source for potential 1 cell replacement therapies endogenously
without the risk of alloreactivity associated with transplantation methods. To suppress
autoreactive T cell responses against newly formed cell mass due to FAKi treatment, we will utilize
the small molecule PFK15 to inhibit glycolysis. Interest in the metabolic programs that dictate T
cell function has gained interest in recent years. Specifically, studies have investigated the
benefit of targeting cellular metabolism as a means to control aberrant T cell activation
phenotypes, especially in the context of autoimmunity. Our recent manuscript and preliminary data
provide evidence that specific targeting of the glycolysis enzyme PFKFB3 with the small molecule
inhibitor PFK15 delays the onset of T1D in a T cell transfer model by inducing CD4+ T cell
exhaustion. The phenotype we observed was irreversible, indicating a terminal differentiation
state. The induction of terminal exhaustion was a permanent phenotype resulting from a short 2-
week treatment, which is beneficial as it would not require life-long administration that could
impact global immune function. We expect use of FAKi and PFK15 to work synergistically as together
they can both restore insulin secretory function all while protecting new cell mass from autoimmune
targeting. Further, both of these therapeutic agents have cleared phase I FDA safety trials and are
ideal candidates to repurpose for clinical use in T1D patients.
Description of Project
Type 1 Diabetes (T1D) is an autoimmune disorder caused by immune-mediated destruction of the pancreatic 1 cell, resulting in insulin deficiency and hyperglycemia. Currently, patients with T1D are treated by the daily administration of exogenous insulin to maintain euglycemia, however, insulin is not a cure. Long-term insulin replacement strategies while effective ultimately puts patients at risk of developing cardiovascular complications, blindness, lower limb amputation, and kidney failure, as well as a reduced life expectancy of approximately 20 years compared to the general population. These factors highlight an urgent need for the identification of novel reversal strategies that can replace lost 1 cell mass while mediating protection of new 1 cells without life- long immunosuppression.
Current therapeutic strategies for T1D have focused on two specific areas: 1) 1 cell replacement or regeneration and 2) immunomodulation. Although innovative efforts have been made to restore 1 cell mass, these strategies ultimately fail due to reemergence of the autoimmune response. Moreover, although immunomodulation has yielded positive results in preclinical studies, success in the clinic has remained limited. Unfortunately, present clinical studies have relied on administering immunotherapies to patients with diagnosed T1D who have suffered significant 1 cell loss. While single- agents alone have failed to prevent or reverse disease, few efforts have attempted to use combinatorial therapies to achieve more successful clinical outcomes by both replacing 1 cell mass while modulating self- reactive T cell responses that protect newly formed/ regenerated insulin- secreting cells. In line with this idea, we have identified novel therapeutic strategies to 1) replace endogenous 1 cell mass by converting pancreatic acinar cells (the most abundant cell type in the pancreas) into insulin-secreting like cells via inhibiting the kinase activity of Focal adhesion kinase (FAK) with the inhibitor PF562271 (termed FAKi); and 2) modulate self- reactive T cell responses by inhibiting the glycolysis enzyme PFKFB3 with the small molecule PFK15. These studies have proven beneficial as single agents in normalizing blood glucose (BG) levels in a streptozotocin (STZ)- induced diabetes model (FAKi) and in preventing adoptive transfer (AT) of diabetes in a model of T1D (PFK15), however whether combinatorial use of FAKi and PFK15 can reverse T1D remains unknown, and is the
basis of our proposal.
Anticipated Outcome
We expect FAKi and PFK15 treatment to reverse T1D onset in vivo by regenerating insulin- secreting
like cells
endogenously from acinar cells (FAKi) and protecting new insulin- secreting cells from autoimmune
targeting by inducing exhaustion of - cell reactive T cells (PFK15). Moreover, we anticipate use of
FAKi and PFK15 as single agents will fail to reverse hyperglycemia in treated animals, therefore
providing proof of concept that combinatorial use of these 2 FDA cleared agents is beneficial in
the reversal of autoimmune diabetes. These studies will provide
precedence for exploring use of this combination clinically.
Relevance to T1D
At present therapeutic intervention for the treatment of T1D has relied on reversing established
disease without
restoring endogenous cell mass. Trials conducted to date were primarily done on patients that were
already diabetic, hoping that resolution of autoimmunity would afford the residual cell mass a
chance, to again normalize blood glucose. Unfortunately, the level of cell mass in this patient
population is insufficient to restore euglycemia and therefore these trials never met endpoints set
forth in the protocols. Although single-agents alone have failed to prevent or reverse disease, few
efforts have attempted to use combinatorial therapies to achieve more successful clinical outcomes
by both replacing 1 cell mass while modulating autoreactive T cell responses that protect newly
formed/ regenerated insulin- secreting cells. We have combined a method for endogenous cell
replacement by repurposing an agent in phase 1 trials, (FAK) inhibitor (FAKi) to replace endogenous
1 cell mass by converting pancreatic acinar cells into insulin-secreting like cells. This provides
endogenous syngeneic like cells to reestablish euglycemia, while overcoming the major alloimmune
barrier common with transplantation methods. In conjunction with this treatment, we will also add
an inhibitor in clinical trials (PFK15) a molecular inhibitor of a rate limiting glycolysis enzyme
known as PFKFB3. Our recent manuscript describes the ability of PFK15 to delay T1D onset via
targeting of the glycolysis pathway. Short term treatment established protection due to induction
of terminal exhaustion, characterized by increased and sustained expression of the checkpoint
molecules PD-1 and LAG-3.
This phenotype was irreversible through restimulation or checkpoint blockade, indicating an ability
to permanently induce T cell exhaustion with a short-term treatment. We are confident that this
preclinical model can be translated to a ready therapy for clinical application as these agents
have cleared phase I FDA clinical trials for cancer.
Successful completion of the proposed specific aims would revolutionize the way we think about
managing and treating T1D. The combinatorial therapy we propose could provide a treatment that
reverses established T1D without the requirements of life- long administration. This would lead to
a better quality of life for affected patients, and reduce the health care costs associated with
autoimmune diabetes.