Prognostic and therapeutic implications of TP53 expression in persistent myelomonocytic leukemia

The tumor suppressor TP53 is probably the most incessantly mutated gene in most cancers [1]. Throughout various myeloid malignancies TP53 disruption is widespread and related to poor-risk illness [2] and therapeutic resistance [3]. Unusually, TP53 mutations are uncommon in persistent myelomonocytic leukemia (CMML) [4, 5], regardless of illness options in any other case overlapping with myelodysplastic syndromes (MDS), myeloproliferative neoplasms and acute myeloid leukemia (AML): every of which harbors sizeable proportions of TP53-mutated instances. Just lately, the most important cohort thus far reported TP53 mutations in solely 2.4% of 1315 CMML instances [4]. Predictably these sufferers displayed adversarial options and inferior AML-free survival (LFS) and general survival (OS) in contrast with TP53 wild-type (TP53^WT) CMMLs.

Whereas the scientific traits of TP53-mutated CMML are actually comprehensively described [4], this represents a small fraction of CMML instances. Moreover, p53 exercise could be modulated by non-mutational mechanisms, for instance by way of altered transcriptional expression, posttranslational modifications, and mobile localization [6]. We hypothesized that p53 dysfunction would possibly in any other case characterize a hitherto-unknown subset of TP53^WT CMML sufferers, and so investigated TP53 mutations, allelic standing, expression degree, and therapeutic response in a big worldwide collaborative CMML cohort.

We studied 648 CMML sufferers from North−West England with out there scientific, mutational, and end result knowledge. Subsets of sufferers handled at The Christie (Manchester, UK) underwent RNA-sequencing on bone marrow (BM) CD34-sorted hematopoietic stem/progenitor cells (HSPCs; n = 33); and p53 immunohistochemical (IHC) staining on archived BM trephine samples (n = 31; n = 14 overlapping each cohorts). Individually, we analyzed 92 sufferers handled at Nationwide Taiwan College Hospital (NTUH, Taipei, Taiwan) for whom presentation BM mononuclear cells (MNCs; n = 92) and RNA-sequencing (n = 90/92) knowledge have been out there [7]. Lastly, we re-analyzed revealed RNA-sequencing knowledge from BM MNCs of 24 sufferers from Hospital Morales Meseguer (Murcia, Spain) [8]. Every cohort included wholesome BM controls (HCs). Extra cohort particulars and experimental strategies are offered as Supplementary Knowledge (Strategies S1−8, Tables S1−3).

Median ages of the 648 UK and 92 Taiwanese sufferers have been 75 and 71 years, respectively, each with male predominance (Tables S4−5). Solely eight (1.23%) and two (2.2%), respectively, carried TP53 alterations (Fig. 1A, B). As anticipated, UK TP53-altered sufferers displayed considerably inferior outcomes in contrast with TP53^WT (Fig. 1A), with an identical development within the Taiwan cohort (Fig. 1B). Thus, we corroborate the paucity of TP53 mutations in CMML [4] throughout beforehand unreported UK and Taiwan cohorts.

Inspecting TP53 gene expression ranges throughout totally different cell sorts in regular and malignant hematopoiesis (from publicly out there datasets) revealed that wholesome HSCs specific considerably larger TP53 than in MDS (p < 0.01, Fig. S1). Subsequently, we explored TP53 expression ranges and their scientific significance in TP53^WT CMML instances, equally observing decrease TP53 within the UK and Spain cohorts (Fig. S2A). Notably, TP53 expression in HSPCs was considerably larger than in MNCs from each wholesome and illness contexts (Fig. S2B).

Our Taiwan RNA-sequencing CMML discovery cohort was stratified into TP53^excessive and TP53^low transcriptional expression teams. TP53^low sufferers displayed considerably decrease expression than HCs, whereas TP53^excessive expression ranges have been similar to controls (Fig. 1C). Thus, whereas most CMML sufferers are TP53^WT, BM cells from a subset (~15%) show abnormally low TP53 expression, suggesting potential for altered p53 (and downstream) perform in these sufferers. Contemplating the purposeful crosstalk between the MDM2-MDMX advanced and p53 we examined correlations between MDMX, MDM2, and TP53. TP53^low expressors exhibited larger MDMX than TP53^excessive, with no distinction noticed for MDM2 (Fig. S3). Scientific and mutational options didn’t differ between the 2 TP53 expression subgroups (Tables S6−11).

Given the affiliation between TP53 mutations and HMA resistance [3], we examined whether or not TP53 expression correlated with HMA response within the TP53^WT context. Regardless of restricted pattern sizes (Desk S12), TP53^low sufferers confirmed a constant development in direction of poorer HMA response charges throughout cohorts (Fig. 1D). TP53^low sufferers displayed considerably shorter LFS and OS than TP53^excessive instances (Fig. 1E). In subgroup analyses, decrease TP53 retained robust predictive worth for LFS and OS even inside CPSS and CPSS-Molecular-stratified subgroups (Figs. S4, 5), and in solely ASXL1^WT sufferers (Fig. 1F). Time-dependent ROC curve evaluation revealed potential for TP53 expression to reinforce present prognostication methods (Fig. 1G). In multivariable evaluation, decrease TP53 expression remained prognostically detrimental for LFS and OS (Desk S13). This was constant throughout validation cohorts (Figs. S6, 7; Tables S14, 15), strengthening the noticed hyperlink between decrease TP53 expression and adversarial outcomes.

We subsequently explored TP53 expression on the protein degree by IHC in 31 CMML trephine samples (Fig. 1H). We discovered no important correlation between p53 IHC and TP53 RNA-sequencing expression ranges: albeit with solely a small overlapping cohort with each out there (Desk S16, 17, Fig. S8), and evaluating totally different populations (complete BM vs CD34 +, respectively). Within the full IHC cohort, nonetheless, low p53 expressing instances exhibited considerably inferior OS (p = 0.048; Fig. 1I), validating our observations evaluating TP53 transcript ranges.

We subsequent sought how decrease TP53 expression would possibly affect CMML biology and prognosis. Concordant with our scientific remark (Fig. 1D), single-sample GSEA confirmed enrichment of HMA resistance signatures in TP53^low sufferers, constantly throughout cohorts (Figs. 2A, S9). We hypothesized that altered TP53 expression is perhaps related to aberrant self-renewal and cell cycle packages: acknowledged mediators of established HMA resistance mechanisms [9]. TP53^low cells confirmed enrichment for LSC and HSC genes, and relative lower in cell cycle-related genes, as in contrast with TP53^excessive and HC (Figs. 2B, S10). Thus, TP53^low CMML shows distinct stemness and quiescence signatures, linked to poor HMA response in these sufferers.

A Field plots displaying resistance signatures derived from single-sample GSEA for hypomethylating brokers (HMA), venetoclax, cytarabine, and daunorubicin in sufferers with decrease and better TP53 expression within the UK CD34 + -sorted cohort. B Field plots displaying scores of leukemic stem cell (LSC), hematopoietic stem cell (HSC), and cell cycle of sufferers with TP53^excessive and TP53^low expression and wholesome controls (HC) within the UK CD34 + -sorted cohort. C Consultant GSEA plots of pathway enrichment in CMML sufferers with the bottom 25% vs highest 25% TP53 expression within the UK CD34 + -sorted cohort. D Violin plots displaying totally different signatures seen within the Taiwan discovery cohort and the UK validation cohort. MEP: megakaryocytic-erythroid progenitors. A, B, D ****P ≤ 0.0001, ***P ≤ 0.001, **P ≤ 0.01, *P ≤ 0.05. P values have been computed utilizing Mann–Whitney or Kruskal–Wallis check. E Consultant 3D synergy plots utilizing zero interplay (ZIP) mannequin (proper) and dose response curves (left) for CMML bone marrow mononuclear cells (n = 3 sufferers; imply + SEM) handled for 72 h ex vivo with NSC-207895 and azacitidine mixture at numerous concentrations. The presence of synergy was decided using the SynergyFinder computational package deal and the ZIP synergy index the place purple denotes synergism and inexperienced denotes antagonism. A optimistic synergy rating is the % extra cell demise than anticipated. F Dot plot displaying the correlation between TP53 expression and synergy rating of 10 affected person samples.

By GSEA TP53^low cells exhibited depleted expression of p53-dependent pathways, together with MYC targets, G2/M checkpoints, and DNA restore (Figs. 2C, S11). Apparently, these have been all among the many most upregulated pathways in TP53-mutant (vs TP53^WT) samples throughout a number of cancers in TCGA knowledge [1], implying that the driving biology of TP53^low CMML is distinct from (and never functionally equal to) that of oncogenic TP53 mutations. Conversely, TP53^low sufferers demonstrated enhanced TNF-alpha and inflammatory alerts (Figs. 2C, S11), highlighting potential crosstalk between p53 and extrinsic components within the CMML BM microenvironment. Comparable outcomes have been noticed in our different cohorts (Fig. S11). Taken collectively, in contrast with HCs or TP53^excessive CMML, TP53^low CMML cells show comparatively quiescent cell cycle however heightened irritation.

With rising proof suggesting discrete roles for p53 in regulating irritation and immune cell panorama [10], we utilized xCell to our transcriptomic datasets to investigate alerts of twenty-two cell sorts. TP53^low CMML displayed considerably stronger M2-macrophage, however decrease megakaryocytic-erythroid progenitors (MEP) alerts in contrast with TP53^excessive (Fig. 2D, Desk S18). Apparently, these findings have been constant within the UK CD34+ dataset, suggesting lineage priming on the progenitor degree.

Lastly, we explored whether or not lowered TP53 expression on this CMML subset could possibly be exploited therapeutically. HMAs are the one accredited disease-modifying medication for CMML however typically yield disappointing responses [11]. Regardless of intensive efforts, no mixture has but reported survival benefit over HMA monotherapy. Because the MDM2/MDMX advanced degrades wild-type p53, twin inhibition might supply extra complete modulation, as recommended by early scientific leads to TP53^WT AML/MDS following HMA failure [12]. Combining NSC-207895, a twin MDMX/MDM2 inhibitor, and p53 activator, with azacitidine at numerous concentrations, we noticed clear and substantial synergy in major samples ex vivo from 10/11 sufferers (Fig. 2E; Desk S19). There was a development in direction of inverse correlation between TP53 expression and empirical synergy scores (Figs. 2F, S12), suggesting potential for pharmacological p53 activation to reinforce HMA sensitivity in CMML with broad efficacy; maybe preferentially in adversarial TP53^low expressing instances (though we couldn’t validate this experimentally, missing availability of matched post-treatment samples).

An intriguing query stays: why are TP53 mutations so rare in CMML? Speculatively, TP53 mutations would possibly induce unknown artificial lethalities in CMML cells; or they could promote various lineage specification pathways, re-directing the expressed phenotype and resultant illness classification. Supporting the latter, most studied TP53^MUT hematopoietic fashions report enhanced stemness or propagation of megakaryocytic/erythroid lineage [13], reasonably than the myelomonocytic expansions that outline the CMML phenotype. For instance, TP53 knockout synergized with NRAS^G12D to particularly remodel MEPs, however not different HSPC sorts, in an AML murine mannequin [14]. Accordingly, we noticed important under-representation of myelomonocytic/blastic M4/M5 FAB subtypes related to TP53 mutations amongst 1511 AML instances at NTUH, and re-analyzing 577 instances from TCGA and BeatAML datasets (odds ratio 0.48 and 0.49, respectively; Desk S20). Thus, acquisition of TP53 mutations onto the canonical CMML mutation background would possibly alter the resultant phenotype away from clinicopathological options suitable with CMML diagnostic standards.

When current, TP53 mutations confer adversarial prognosis in CMML as in different cancers. Nonetheless, our research identifies prognostic implications of mutation-independent TP53 dysregulation in CMML related to a a lot bigger minority of sufferers (~15%). Prior TCGA evaluation revealed substantial variation in TP53 expression in each TP53^MUT and TP53^WT tumors [15], with TP53^WT expression decrease than in missense however larger than in truncating mutations. Moreover, the connection between expression and prognosis differed throughout cancers [15]. Our knowledge counsel that in CMML TP53 expression degree performs a job in dictating illness aggressiveness and therapeutic response, of comparatively larger significance than TP53 mutation standing on this illness.

In conclusion, ours is the primary research to hyperlink low TP53 expression with distinct options and outcomes in CMML. We verify the rarity of TP53 mutations, while figuring out a novel subgroup with aberrantly low TP53 expression, related to larger HMA resistance, distinctive biology, and inferior prognosis. We spotlight potential for combining HMA and MDMX/MDM2 inhibition to revive HMA sensitivity, as a sexy candidate therapeutic method for scientific research to deal with this unmet scientific want.