Novel AML Strategies Aim to Restore Regulatory Gene Functions

OncologyLive, Vol. 20/No.4, Volume 20, Issue 4

Acute promyelocytic leukemia, a rare genetic subtype of de novo acute myeloid leukemia is treated not with conventional cytotoxic chemotherapy but with just 2 drugs: all-trans retinoic acid and arsenic trioxide. Given this, an enduring question has been whether noncytotoxic differentiation-restoring therapy can be extended to the most common genetic subtypes of AML.

Yogen Saunthararajah, MD

Hematology/Medical Oncology Specialist

Department of Translational Hematology and Oncology Research

Taussig Cancer Institute

Cleveland Clinic

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.

Moreover, use of the clinical small molecule decitabine (Dacogen) to directly deplete the corepressor DNMT1 from the CEBPA/RUNX1 interactomes also reconfigured CEBPA/RUNX1 interaction from corepressors to coactivators, which activated terminal granulocytic fates. Interestingly, as per normal granulocytic differentiation, this led to a natural downregulation of NPM1 (including mutant NPM1) protein.

In a patient-derived xenotransplant model of poor-prognosis dual NPM1/FLT3-mutated AML, with treatment initiated after confirmation of bone marrow engraftment to greater than 20% AML cells, combination noncytotoxic therapy with pharmacodynamically directed dosing of selinexor and DNMT1-depleting drugs extended time-to-distress by more than 6 months over vehicle treatment. The noncytotoxic mechanism of action was highlighted by the preservation of normal blood counts during more than 8 months of active therapy with the regimen. The NPM1/FLT3-mutated AML cells that resisted and progressed through several months of in vivo therapy to cause distress in the mice did so by avoiding both nuclear retention of mutant NPM1/PU.1 by selinexor and DNMT1-depletion by decitabine/ 5-azacytidine. That is, resistance both in vitro and in vivo was by prevention of intended molecular pharmacodynamic effects, underscoring the importance of the targeted pathways to the malignant phenotype. A summary of the data is provided (Figure 2).

PU.1, CEBPA, and RUNX1 have been shown to promote exponential replication kinetics by binding to MYC enhancers to produce high-grade activation of MYC (the master transcription factor coordinator of cell proliferation) and cobinding with MYC at its target genes. This contrasts with the quiescence imposed by stem cell master transcription factors such as HLF in hematopoietic stem cells. In computational analyses, such skewing of intrinsic replication rates logarithmically favors decoupling replication from forward differentiation in lineage progenitors as the most efficient strategy for malignant transformation. The results demonstrated how mutant NPM1 executes such decoupling in lineage progenitors and showed how the machinery could be recoupled.

The investigators do not know for sure why neoplastic evolution selects to mutate NPM1 instead of PU.1 directly, but possible reasons include that mutant NPM1 creates the graded PU.1 loss of function (more than haploinsufficiency, less than total) that has been shown to be necessary for leukemogenesis. It’s also possible that dislocation of proteins other than PU.1 contributes to transformation. The leukemogenic suspension of cells at an intermediate, inherently replicative stage of their advance along lineage-differentiation axes seems to hinge on partial but not complete loss of function in the PU.1/ CEBPA/RUNX1 circuit. This is because, experimentally, complete inactivation of PU.1, CEBPA, or RUNX1 kills AML cells, even as partial loss of function of any 1 of these is leukemogenic; accordingly, NPM1, RUNX1, and biallelic CEBPA mutations, though highly recurrent in AML, are mutually exclusive.

This delicate balancing act of all AMLs, relying on commanders of lineage differentiation and being poised close to the edge of terminal myeloid differentiation, is what the investigators fundamentally exploit with their evaluated treatments. These results add to a body of data suggesting that corepressor-coactivator imbalance in the PU.1/CEBPA/RUNX1 master transcription factor hub is a common final pathway that represses terminal differentiation. This explains the meaningful clinical activity of noncytotoxic DNMT1 depletion by decitabine (or its prodrug 5-azacytidine) in patients with myeloid malignancies containing sundry mutations and translocations.4

Therefore, mutant NPM1 disrupts the PU.1/CEBPA/ RUNX1 master transcription factor hub to stall myeloid differentiation at an inherently proliferative stage of myeloid lineage differentiation. By dissecting this final common pathway to myeloid differentiation arrest and leukemic hematopoiesis, the investigators identified and then validated specific molecular targets for noncytotoxic (p53-independent, normal stem cell sparing), differentiation-restoring therapy of the largest genetic subtypes of AML. Because the treatments have defined and measurable molecular pharmacodynamic effects, dosages of candidate therapeutics can be selected rationally instead of empirically, and measurements for achievement of intended molecular pharmacodynamic effects can facilitate rational investigation of mechanisms of resistance. In sum, the discoveries regarding mutant NPM1 can guide efforts to extend noncyotoxic (nontoxic) differentiation-restoring treatments to the most common genetic subset of AML.


  1. Gu X, Ebrahem Q, Mahfouz RZ, et al. Leukemogenic nucleophosmin mutation disrupts the transcription factor hub regulating granulomonocytic fates. J Clin Invest. 2018;128(10):4260-4279. doi: 10.1172/JCI97117.
  2. Rapin N, Bagger FO, Jendholm J, et al. Comparing cancer vs normal gene expression profiles identifies new disease entities and common transcriptional programs in AML patients. Blood. 2014;123(6):894-904. doi: 10.1182/blood-2013-02-485771.
  3. Bagger FO, Sasivarevic D, Sohi SH, et al. BloodSpot: a database of gene expression profiles and transcriptional programs for healthy and malignant haematopoiesis. Nucleic Acids Res. 2016;44(D1):D917-924. doi: 10.1093/nar/gkv1101.
  4. Velcheti V, Schrump D, Saunthararajah Y. Ultimate precision: targeting cancer but not normal self-replication. Am Soc Clin Oncol Educ Book. 2018;(38):950-963. doi: 10.1200/EDBK_199753.