Presented at the STLCOP Research Symposium in the Spring of 2015.

1. THERAPEUTIC STRATEGIES AIMED TO CIRCUMVENT P-GLYCOPROTEIN-MEDIATED
RESISTANCE IN MULTIPLE MYELOMA
Nicholas Potter1,2
, Pharm.D. Candidate 2017, Benjamin Minch1,2
, Pharm.D. Candidate 2017, Barbara Muz2
,Ph.D., Noha N. Salama1
, Ph.D., and Abdel Kareem Azab2
,Ph.D.
1
St. Louis College of Pharmacy, Department of Pharmaceutical and Administrative Sciences, St. Louis, MO.
2
Washington University Medical School, Department of Radiation Oncology, St. Louis, MO.
ABSTRACT
Introduction: Multiple myeloma (MM) is a malignant neoplastic cancer of the
plasma cells that involves the bone marrow (BM). Generally, patients respond
initially to treatment, but become refractory later-on. With novel therapeutic
agents, the median survival of MM patients is estimated to be about 7 years;
however, patients that are refractory to these drugs (such as proteasome
inhibitors) have an overall average survival of 9 months. Hypoxia develops in
the BM niche during MM progression and plays a major role in MM cell
dissemination to new BM niches. Additionally, hypoxia has long been linked to
chemoresistance. Hypoxia-Inducible Factor (HIF-1α) is upregulated in many
hypoxic cells, and is the key regulator of a cell’s hypoxic response. Increased
expression of efflux proteins, such as P-glycoprotein (P-gp), is a major reason
for drug resistance in MM because many MM drugs are P-gp substrates (such
as proteasome inhibitors). MDR1, the gene encoding P-gp, is a target gene of
HIF-1α. We hypothesize that inhibition of the hypoxic response in MM cells by
inhibition of the HIF-1α pathway will prevent P-gp induction and re-sensitize
MM cells to therapy. We explored two strategies to inhibit or minimize the
development of P-gp mediated drug resistance in MM: 1) a direct approach
through targeting P-gp efflux using a P-gp inhibitor and 2) an indirect
approach through targeting the genetic regulation of HIF-1α .
Methods: MM1s cells (n =3/treatment), MM cell line, were incubated under
normoxic and hypoxic conditions for 24 hrs. with 1) Tariquidar, a P-gp
inhibitor, 2) Px-478, a HIF-1α inhibitor or 3) Tariquidar and Px-478. The cells
were then treated with Rhodamine 123 (Rh123, a fluorescent model P-gp
substrate) for 1 hr. at 1 ug/mL. The intracellular Rh123 concentration was
measured by flow cytometry and compared statistically using ANOVA and
Bonferroni pairwise comparisons at P<0.05 (SPSS 20).
Results: We did not detect a significant difference in the cellular Rh123
content in the absence or presence of any agent under normoxic conditions.
However, under hypoxic conditions, incubation of the cells with either
Tariquidar or Tariquidar/Px-478 caused a significant increase in the amount of
Rh123 accumulating inside the cells. When comparing the effect of hypoxia to
normoxia on Rh123 accumulation intracellularly with and without inhibitors,
there was a substantially lower (P <0.05) Rh123 intracellular content in MM1s
cells incubated under hypoxic conditions in the absence and presence of Px-
478. In contrast, the extent of Rh123 intracellular accumulation following
treatment of the cells with Tariquidar or Tariquidar/Px-478 was not significantly
different under normoxic and hypoxic conditions.
Conclusions: The extent of Rh123 intracellular accumulation is a measure of
P-gp efflux. In this study, we detected an increase in the extent of Rh123
effluxed out of the MM1s cells under hypoxic conditions, which suggests P-gp
upregulation and is consistent with recent studies reported by our research
group. HIF inhibition was recently reported to be a successful strategy to
prevent P-gp upregulation in hypoxia and subsequently interfere with drug
resistance in other types of cancer. However, in our study, HIF-1α inhibition
had no effect on Rh123 efflux under hypoxic conditions. The co-incubation of
Tariquidar with Px-478 did not provide an enhanced effect beyond that
observed with Tariquidar incubation alone. Future studies will focus on
exploring higher concentrations of Px-478 as well as other HIF-1α inhibitors.
On the contrary, inhibition of P-gp-mediated Rh123 efflux by Tariquidar had no
effect in normoxia (limited or no P-gp expression) but significantly increased
the extent of intracellular Rh123 accumulation under hypoxic conditions where
P-gp is upregulated. Hence, P-gp inhibition proved to be a successful strategy
to interfere with the development of P-gp mediated drug resistance in MM.
CONCLUSIONS
Our research group reported enhanced MM drug resistance to
current therapies when MM cells are hypoxic.3
In the study
described herein, we used the accumulation of the P-gp substrate
Rh123 as an indicator of the change in P-gp activity in cells under
hypoxic versus normoxic conditions. We documented a 43.4%
decrease in Rh123 intracellular content under hypoxia compared
to normoxic conditions, which is comparable to earlier findings.9
Because HIF-1α targets MDR1 (the gene encoding P-gp) we
expected that inhibition of HIF-1α would restore Rh123
intracellular accumulation under hypoxic conditions. However,
using Px-478 at 25uM did not prove effective in minimizing P-gp-
mediated drug resistance under hypoxia in the tested MM cell
lines. Future studies will explore additional or alternative specific
HIF-1α inhibitors at a range of concentrations. The extent of
contribution of other cellular mechanisms of drug resistance to the
decline in Rh123 intracellular content remains elusive.
On the contrary, inhibition of P-gp via Tariquidar under hypoxic
conditions restored the extent of Rh123 intracellular accumulation
to the levels detected under normoxia. Combining both Px-478
and Tariquidar provided no synergistic effects when compared to
Tariquidar treatment alone. This observation suggests that
Tariquidar was the sole contributor to the restoration of Rh123
cellular levels and hence, P-gp inhibition is an effective strategy to
prevent the development of resistance to P-gp substrates such as
the MM drugs, Carfilzomib, Doxorubicin, and Melphalan in
hypoxia-induced resistant MM cells lines.
REFERENCES
1.Abraham J et al. The role of P-glycoprotein in drug resistance in multiple
myeloma. Leuk Lymphoma. 2015;56(1):26-33.
2.Azab A et al. Hypoxia promotes dissemination of multiple myeloma through
acquisition of epithelial to mesenchymal transition-like features. Blood.
2012;119(24):5782-5794.
3.Muz B et al. Hypoxia promotes stem cell-like phenotype in multiple myeloma
cells. Blood Cancer Journal. 2014;4(12):e262.
4.De la Puente P et al. Molecularly Targeted Therapies in Multiple
Myeloma. Leukemia Research and Treatment. 2014;2014:976567.
5.Welsh Set al. Antitumor activity and pharmacodynamic properties of PX-478, an
inhibitor of hypoxia-inducible factor-1alpha. Mol Cancer Ther. 2004;3(3):233-44.
6.Thomas H and Coley H. Overcoming multidrug resistance in cancer: an update
on the clinical strategy of inhibiting P-glycoprotein. Cancer Control. 2003;10(2):159-
65.
7.Mistry P et al. In vitro and in vivo reversal of P-glycoprotein-mediated multidrug
resistance by a novel potent modulator, XR9576. Cancer Res. 2001;61:749-758
8.Chen J et al. HIF-1α inhibition reverses multidrug resistance in colon cancer cells
via downregulation of MDR1/P-Glycoprotein. Zmijewski M, ed. PLoS ONE.
2014;9(6):e98882.
9.Abraham J et al. Hypoxia induces up-regulation of p-glycoprotein and promotes
resistance to carfilzomib in multiple myeloma. AAPS Annual Meeting and
Exposition, November 2-6, 2014, San Diego, CA.
BACKGROUND
Multiple Myeloma (MM) is a type of malignant cancer of bone
marrow plasma cells. There are 20,000 new cases yearly with
10,000 deaths attributed to this disease.1
The median survival for patients with MM is about 4-5 years with
advancements to 7 years with newer therapy.1
This low survival
rate is attributed to the development of resistance mechanisms
that render MM unresponsive to previously efficacious
therapies.1
Regions of low blood concentration (hypoxia) arise in cancerous
tumors due to enhanced cell activity and division, coupled with
insufficient angiogenesis.2
Hypoxia is also a desirable
therapeutic target for MM as it has a potentially high therapeutic
index.
Several studies have concluded that hypoxia promotes cancer
cell progression and survival through various transcription
factors.2
Azab, et al. reported enhanced MM drug resistance to
current therapies when MM cells are hypoxic.3
Hypoxia-Inducible Transcription Factor-1 (HIF-1) is a
heterodimer; consisting of the HIF-1α and HIF-1β subunits, with
HIF-1α playing a major role in a cell’s hypoxic response.4
Studies have demonstrated that the inhibition of HIF-1α blocks
MM angiogenesis, tumor burden, and bone destruction in vivo.4
Px-478 is reported to be an effective HIF-1α inhibitor in many
forms of cancer.5
P-glycoprotein (P-gp) is a drug transporter, induced by the
MDR1 gene1
. It effluxes its substrates from inside the cell to the
extracellular space. Treatment with chemotherapeutic agents
(e.g. doxorubicin, carfilzomib) was associated with P-gp
upregulation in MM cells, leading to decreased drug efficacy.
Tariquidar is a third-generation P-gp inhibitor that non-
competitively binds with high affinity to this efflux pump.6
It was
shown to inhibit P-gp activity for 22 hours in vitro after the drug
was removed from the culture medium.7
MDR1, the gene encoding P-gp, is a target gene of HIF-1α.
Recent studies involving colon cancer reported that HIF-1α
inhibition reverses drug resistance via downregulation of
MDR1/P-gp.8
A similar correlation has not been confirmed in
MM.
HYPOTHESIS
We hypothesize that inhibition of the hypoxic response in MM
cells by inhibition of the HIF-1α pathway will prevent P-gp
induction and re-sensitize MM cells to therapy.
OBJECTIVES
We explored two strategies to inhibit the development of P-gp
mediated drug resistance in MM: 1) a direct approach through
targeting P-gp efflux using a P-gp inhibitor and 2) an indirect
approach through targeting the genetic regulation of HIF-1α.
METHODS
MM1s cell line was used as a biological model for MM. The cells
(5x 105
cells/ well) were incubated for 24 hours under either
normoxic (37 o
C, 5% CO2, 21% O2) or hypoxic (37 o
C, 5% CO2,
1% O2) conditions with/without one of the following treatments:
1)RPMI 1640 growth medium with L-glutamine
2)Px-478 (25 uM, HIF-1α inhibitor) in RPMI 1640 medium
3)Tariquidar (1 uM, P-gp inhibitor) in RPMI 1640 medium
4)Px-478 (25uM) and Tariquidar (1uM) in RPMI 1640 medium
The fluorescent P-gp substrate; Rhodamine 123 (Rh123,
1ug/mL) was subsequently incubated with the cells for 1 hour.
The MM1s cells were then washed 3 times by PBS 1x.
The mean fluorescence intensity (MFI) for the intracellular Rh123
was measured by flow cytometry.
Control wells (without Rh123) were used to measure the
background/noise.
Statistical analysis: The different treatments were compared
statistically using ANOVA and Bonferroni pairwise comparisons
at P<0.05 (SPSS 20).
RESULTS
Under normoxic conditions, we did not detect a significant
difference between the intracellular concentrations of Rh123
under any treatment in normoxic conditions. The untreated, Px-
478-treated, Tariquidar-treated, and Px-478/Tariquidar- treated
cells showed intracellular Rh123 concentrations of 2.71 ± 0.16,
2.31 ± 0.20, 2.30 ± 0.07, and 2.42 ± 0.38 respectively (p>0.05).
Under hypoxia, Rh123 intracellular concentrations were not
significantly different when comparing Px-478 treated cells to the
control untreated cells.
RESULTS
The intracellular concentrations of Rh123 in Tariquidar-treated
and Px-478/Tariquidar-treated hypoxic cells were not statistically
different from each other. However, their Rh123 intracellular
content was significantly (p<0.05) higher than the levels
detected with the untreated and Px-478 treated cells.
When comparing incubation under hypoxic and normoxic
conditions, we detected a significant decrease (43.4%) in Rh123
intracellular concentrations in untreated hypoxic cells (1.89 ±
0.08) compared to untreated normoxic cells (2.71 ± 0.16). This
observation was reported earlier by our research group.9
Similarly, treatment of the cells with Px-478 resulted in
significantly lower (22.9%) Rh123 intracellular content in hypoxic
cells (1.88 ± 0.13) compared to normoxic cells (2.31 ± 0.20).
On the contrary, treatment of the cells with Tariquidar or with Px-
478/Tariquidar did not show any substantial differences in
Rh123 intracellular content under hypoxic versus normoxic
conditions.
Intracellular Rh123 Concentrations Under Hypoxia
Untreated Px-478 Tariquidar Px-478/Tariquidar
1.89 ± 0.08
(100%)
1.88 ± 0.13
(99.5%)
2.35 ± 0.16 *
(124.3%)
2.53 ± 0.08 *
(133.9%)
* p<0.05
ACKNOWLEDGEMENTS
This research was supported by the Washington University
Institute of Clinical and Translational Sciences grant
UL1TR000448 from the National Center for Advancing
Translational Sciences (NCATS) of the National Institutes of
Health (NIH). The content is solely the responsibility of the
authors and does not necessarily represent the official view of
the NIH. Additional resources were provided through Federal
work Study and Departmental Research Assistantship Funds.
Fig 1. The intracellular Rhodamine 123 concentrations in
MM1s cells, in the presence and absence of P-gp and HIF-1α
inhibitors under hypoxic and normoxic conditions.
0
20
40
60
80
100
120
Control PX-478 Tariquidar PX-478/Tariq
IntracellularcontentofRh123
(%ofuntreatedcomtrolMM1s)
Normoxia
Hypoxia
**
* p<0.05