Novel AML Strategies Aim to Restore Regulatory Gene Functions

Yogen Saunthararajah, MD
Published: Wednesday, Feb 13, 2019
Wake ForestYogen Saunthararajah, MD
Yogen Saunthararajah, MD
 
Hematology/Medical Oncology Specialist
Department of Translational Hematology and Oncology Research
Taussig Cancer Institute
Cleveland Clinic
Strategic Partnership
The disseminated malignancy with the best overall survival today is acute promyelocytic leukemia (APL), a rare genetic subtype of de novo acute myeloid leukemia (AML), which accounts for less than 10% of AML incidence. It is treated not with conventional cytotoxic chemotherapy but with just 2 drugs: all-trans retinoic acid and arsenic trioxide. These unblock the myeloid differentiation arrest that defines all AMLs and release the APL cells to terminal granulocyte lineage fates. Given this inspiring example of what is clinically possible, an enduring question has been whether noncytotoxic differentiation-restoring therapy can be extended to the most common genetic subtypes of AML—eg, those containing mutated nucleophosmin (NPM1), which amount to approximately 30% of AML cancers.

The master transcription factor expression pattern of AML cells suggests why this should be achievable. A few of the roughly 100 transcription factors expressed in cells are master regulators, collaborating in couplets or triplets to powerfully determine cell fates and functions, as illustrated by their remarkable capacity to convert cells of one lineage into another—even into embryonic stem cells.

The master regulators highly expressed in all AMLs are PU.1, CEBPA, and RUNX1, which usually collaborate to command terminal granulocyte or monocyte lineage fates (Figure 1).1-3 Clearly, AML cells evade such terminal lineage fates, but how do they defy these master regulators, and can compliance be restored?

Historically, investigators have known that translocations of NPM1 cause cytoplasmic dislocation of the NPM1 protein, but why or how this should transform myeloid cells was unknown. The Cleveland Clinic group performed the first mass-spectrometric analyses of protein–protein interactions of endogenous NPM1 affinity purified from wild-type and NPM1-mutated AML cell nuclear and cytoplasmic fractions. They found that both wild-type and mutant NPM1 interact with PU.1 and that mutant NPM1 dislocates PU.1 into the cytoplasm.

CEBPA and RUNX1—master transcription factors that collaborate with PU.1 to activate granulomonocytic lineage fates—remained nuclear; but without PU.1, their coregulator interactions were toggled from coactivators to corepressors, suppressing instead of activating more than 500 granulocyte and monocyte terminal-differentiation genes. The PU.1 dislocation also explained why the AML cells expressed high levels of key precursor genes—eg, HOXA9—since these are usually repressed by PU.1-helmed forward myeloid differentiation.

Protein macromolecules such as NPM1 require transport factors to enter (importins) and exit (exportins) the nucleus. Use of selinexor, an inhibitor of XPO1-mediated nuclear export, locked mutant NPM1/PU.1 in the nucleus and activated terminal monocytic fates. The same treatment did not induce differentiation of NPM1-wild type AML cells.

Investigators then queried whether the master transcription factors CEBPA and RUNX1, which remained nuclear in NPM1-mutated AML cells, could be used for a complementary approach to differentiation restoration. CEBPA and RUNX1 are supposed to activate granulocytic fates; however, the granulocyte differentiation program, like the monocyte differentiation program, is suppressed in AML. To investigate how, investigators examined the coregulator interactions of nuclear CEBPA and RUNX1 in NPM1-mutated AML cells using affinity purification–mass spectrometry/Western blotting. The CEBPA and RUNX1 protein interactomes were enriched for coregulators that repress transcription (eg, DNMT1, NURD) over coactivators that activate genes (eg, SWI/SNF, NUA4). PU.1 introduction into the CEBPA/RUNX1 interactomes by selinexor switched CEBPA and RUNX1 interaction in NPM1- mutated AML cells from corepressors to coactivators. This outcome reinforced previous findings from the group regarding how PU.1 and RUNX1 collaboratively exchange corepressors for coactivators. This occurred through interactions between their transcriptionregulating domains.


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