**Abstract**

New drugs targeting single mutations have been recently approved for Acute Myeloid Leukemia (AML) treatment, but allogeneic transplant still remains the only curative option in intermediate and unfavorable risk settings, because of the high incidence of relapse. Molecular analysis repertoire permits the identification of the target mutations and drives the choice of target drugs, but the etherogeneity of the disease reduces the curative potential of these agents. Primary and secondary AML resistance to new target agents is actually an intriguing issue and some of these mechanisms have already been explored and identified. Changes in mutations, release of microenvironment factors competing for the same therapeutic target or promoting the survival of blasts or of the leukemic stem cell, the upregulation of the target-downstream pathways and of proteins inhibiting the apoptosis, the inhibition of the cytochrome drug metabolism by other concomitant treatments are some of the recognized patterns of tumor escape. The knowledge of these topics might implement the model of the 'AML umbrella trial' study through the combinations or sequences of new target drugs, preemptively targeting known mechanisms of resistance, with the aim to improve the potential curative rates, expecially in elderly patients not eligible to transplant.

**Keywords:** acute myeloid Leukemia, FLT3 inhibitors, IDH inhibitors, BCL2 inhibitors, mechanisms of resistance, immunotherapy, target therapy

#### **1. Introduction**

The better knowledge of leukemogenesis has led in the last few years to approval of new target drugs for AML treatment. The availability of these drugs has dramatically changed the AML treatment guidelines, supported by the evidence of their efficacy on a molecular driven basis approach. Neverthless primary resistance and clonal evolution leading to adaptive resistance is a recurring theme even in this setting.

Actually acute myeloid leukemia (AML) is the result of a multi-step sequence of events resulting in impairment of lineage differentiation, hematopoiesis and enhanced self-renewal. Somatic mutations contribute to AML pathogenesis in different manner. Analysis of healthy population exomic and genomic sequencing [1] showed a correlation between pre-leukemic somatic mutations (IDH1/2, SRSF2, U2AF1, TP53, RUNX1, PPM1D) and subsequent development of AML, as first step

process towards leukemogenesis. The subsequent acquisition of mutations appeared to be related with different AML phenotypes. The Cancer genome atlas research network [2] identified eight different genetic pathways responsable of leukemogenesis in 200 adult patients, shown in **Table 1** (trancriptor factor genes fusion and hyperexpression; nucleophosmin 1 delocalization; tumor suppressor genes inhibition; mutations of: DNA-methylation related genes, activated signaling genes, chromatinmodifying genes, cohesin-complex genes, spliceosoma-complex genes). Afterwards Papaemmanuil et al. [3] identified three other molecular subgroups including: IDH2R172 mutation in 1% of AML, mutually exclusive with NPM1, associated with more severe alterations of metabolic activity in comparison to other IDH2 mutations; CCAAT/enhancer binding protein alpha (CEBPA) biallelic mutated AML and inv3 or t(3;3) AML with MECOM (EVI1) and GATA2 mutations. Furthermore Ibanez et al. [4] analyzed 100 patients with normal karyotype AML, lacking NPM1, FLT3, and CEBPA mutations, identifying thirteen seed-genes involved in leukemogenesis with a mean of 4.89 mutations per sample. The network analysis showed a high heterogeneity of gene mutations in this setting and suggested that a specific alteration could not be essential for leukemogenesis, as the interaction between several deregulated pathways.


*\* Class 1 and 7 mutations are both included in the category of mutations of transcription factors genes.*

**289**

**Figure 1.**

*Mechanisms of Resistence of New Target Drugs in Acute Myeloid Leukemia*

• find the correlation with mutations and subclonal architecture;

recent approval of FLT3, BCL2 and IDH inhibitors (FLT3i, BCL2i, IDHi).

*FLT3 pathway (green label) and mechanisms of resistance to FLT3i (light yellow labels).*

We briefly resume the mechanisms of leukemogenesis addressed by these drugs. FLT3 tirosin kinase receptor mutations determines the constitutive activation and dimerization status of the receptor itself, indipendently from FLT3 ligand binding, and the downstream activation of leukemic cells prolipheration and pro-survival pathways (RAS-NFKB, JAK–STAT, PI3K, BCL2) as showed in **Figure 1** [5]. BCL2 is an antiapoptotic protein of BCL2 family which compete with

The perspective of the comprehension of the eterogeneity of the disese inspired recent studies exploring genetic and transcriptomic single leukemic cell analysis

• define a hierarchies of leukemic clones, compared to normal hemapotoiesis;

• identify new markers and leukemic stem cell (LSC) specific gene repertoire.

The aknowledgement of these data will promote the finding of future targets for the eradication of the disease even in the biologically chemoresistant setting of LSC. Uptoday the understanding of leukemogenesis mechanisms have led to the

*DOI: http://dx.doi.org/10.5772/intechopen.94978*

with the following aims:

#### **Table 1.**

*Cathegories of AML mutations and their role in leukemogenesis.*

*Acute Leukemias*

pathways.

Transcription deregulation and impaired hematopoietic

Aberrant localizationn of NPM1 and MPM1-interacting protein.

transcriptional deregulation and impaired degradation through the mouse double minute 2 homolog (MDM2) and the phosphatase and tensin homolog (PTEM).

2-hydroxyglutarate (2HG) which deregulates DNA methylation

Proliferative advantage through the RAS– RAF, JAK–STAT, and PI3K– AKT signaling pathways.

Epigenetic modification and accumulation of

Deregulation of chromatin modification through methylation of histone or impairment of DOT1L (DOT1-like histone H3K79

Chromosome segregation and transcriptional regulation.

methyltransferase).

differentation.

process towards leukemogenesis. The subsequent acquisition of mutations appeared to be related with different AML phenotypes. The Cancer genome atlas research network [2] identified eight different genetic pathways responsable of leukemogenesis in 200 adult patients, shown in **Table 1** (trancriptor factor genes fusion and hyperexpression; nucleophosmin 1 delocalization; tumor suppressor genes inhibition; mutations of: DNA-methylation related genes, activated signaling genes, chromatinmodifying genes, cohesin-complex genes, spliceosoma-complex genes). Afterwards Papaemmanuil et al. [3] identified three other molecular subgroups including: IDH2R172 mutation in 1% of AML, mutually exclusive with NPM1, associated with more severe alterations of metabolic activity in comparison to other IDH2 mutations; CCAAT/enhancer binding protein alpha (CEBPA) biallelic mutated AML and inv3 or t(3;3) AML with MECOM (EVI1) and GATA2 mutations. Furthermore Ibanez et al. [4] analyzed 100 patients with normal karyotype AML, lacking NPM1, FLT3, and CEBPA mutations, identifying thirteen seed-genes involved in leukemogenesis with a mean of 4.89 mutations per sample. The network analysis showed a high heterogeneity of gene mutations in this setting and suggested that a specific alteration could not be essential for leukemogenesis, as the interaction between several deregulated

**Mechanisms of action Class of mutations Mutations/translocations** 

fusions\*

genes

genes

genes

genes

genes

*Class 1 and 7 mutations are both included in the category of mutations of transcription factors genes.*

factor genes\*

Class 8 Cohesin-complex

Transcription deregulation Class 7 Myeloid transcription

Deregulation of RNA processing. Class 9 Spliceosoma-complex

*Cathegories of AML mutations and their role in leukemogenesis.*

Class 1 Transcription factor

Class 3 Tumor suppressor

Class 4 DNA-methylationrelated genes: DNA hydroxymethylation

Class 5 Activated signaling

Class 6 Chromatin-modifying

**(prevalence)**

fusions (18%)

Class 2 NUCLEOPHOSMIN 1 NPM1 mutations (27%)

t(8;21), t(16;16), t(15;17), MLL

TP53, WT1, PHF6 (16%)

TET2, IDH1, IDH2, DNA methyltransferases DNMT3A

FLT3, KIT, RAS mutations

ASXL1, EZH2 mutations, MLL fusions, MLL partial tandem duplications (30%)

CEBPA, RUNX1 mutations

STAG2, RAD21, SMC1, SMC2

SRSF2, U2AF3S, ZRSR2 (14%)

(44%)

(59%)

(22%)

(13%)

**288**

*\**

**Table 1.**

The perspective of the comprehension of the eterogeneity of the disese inspired recent studies exploring genetic and transcriptomic single leukemic cell analysis with the following aims:


The aknowledgement of these data will promote the finding of future targets for the eradication of the disease even in the biologically chemoresistant setting of LSC.

Uptoday the understanding of leukemogenesis mechanisms have led to the recent approval of FLT3, BCL2 and IDH inhibitors (FLT3i, BCL2i, IDHi).

We briefly resume the mechanisms of leukemogenesis addressed by these drugs. FLT3 tirosin kinase receptor mutations determines the constitutive activation and dimerization status of the receptor itself, indipendently from FLT3 ligand binding, and the downstream activation of leukemic cells prolipheration and pro-survival pathways (RAS-NFKB, JAK–STAT, PI3K, BCL2) as showed in **Figure 1** [5]. BCL2 is an antiapoptotic protein of BCL2 family which compete with

*FLT3 pathway (green label) and mechanisms of resistance to FLT3i (light yellow labels).*

BH3 for the binding with the pro-apoptotic proteins BAK/BAX [6]. It inhibits the BH3-BAK/BAX domain and its interaction with the mitochondrial membrane, blocking the p53 dependent mitochondrial apoptosis pathway of the leukemic cell (**Figure 2**). Isocitrate dehydrogenases are cytoplasmic (IDH1) and mitochondrial (IDH2) enzymes cathalyse the reduction of a-ketoglutarate (a-KG) to citrate in krebs cycle in a NADPH-dependent way. NADPH is important for the reduction of glutathione, which in the reduced state is a major antioxidant and protects the cell against reactive-oxygen species (ROS) and other free radicals. IDH mutations have a loss of function effect, producing the accumulation of the oncometabolite R2-hydroxyglutarate (2-HG) which competitively inhibits multiple α-ketoglutarate dependent dioxygenases such as lysine (K)-specific demethylase (KDM) and ten eleven translocation methylcytosine dioxygenase 2 (TET2), causing widespread epigenetic changes with global dysregulation of gene expression and abnormal differentiation and proliferation of leukemic cells (**Figure 3**) [7]. Furthermore 2-HG activates the EglN family of prolyl 4-hydroxylases (EglN), with consequent ubiquitination and degradation of HIF1a, impairing p53 dependent apoptosis. IDH1

**291**

**Figure 3.**

*yellow label).*

*Mechanisms of Resistence of New Target Drugs in Acute Myeloid Leukemia*

mutations also result in a lack of crucial metabolites including a decrease in the NADPH pool and inhibition of krebs cycle with metabolic changes conferring chemotherapy resistance of leukemic cell. At last 2-HG determines a leukemic status highly BCL-2 dependent, preventing the hypoxia mediated apoptosis, determined

*Mechanism of leukemogenesis of IDH mutations (green label) and mechanisms of resistance to IDHi (light* 

Recent studies utilizing NGS and single-cell technologies have also illustrated the complex and polyclonal nature of resistance to targeted therapeutics including FLT3, BCL2 and IDH inhibitors (FLT3i, BCL2i, IDHi) [8, 9]. Here we report the results of the principle studies aiming to analyze mechanism of primary and

FLT3 is a Tirosin Kinase receptor expressed by hematopoietic progenitors and mutated in 25-30% AML. The mutations involve two different domains: the iuxtamembrane domain (FLT3 ITD) in 20-25% AML and the tirosin kinase domain (TKD) in 5-10% AML, expecially at codon D835. They both determine the constitutive activation of the FLT3 receptor tyrosine kinase, inducing cellular proliferation and survival and inhibiting differentiation, through the activation of PI3K/AKT/

secondary leukemic resistance to new approved target therapies.

mTOR pathway, with a critical role in leukemogenesis [10] (**Figure 1**).

*DOI: http://dx.doi.org/10.5772/intechopen.94978*

by cytochrome c oxidase inhibiton.

**2. New target therapies in AML**

**2.1 FLT3 inhibitors (FLT3i)**

*Mechanisms of Resistence of New Target Drugs in Acute Myeloid Leukemia DOI: http://dx.doi.org/10.5772/intechopen.94978*

#### **Figure 3.**

*Acute Leukemias*

BH3 for the binding with the pro-apoptotic proteins BAK/BAX [6]. It inhibits the BH3-BAK/BAX domain and its interaction with the mitochondrial membrane, blocking the p53 dependent mitochondrial apoptosis pathway of the leukemic cell (**Figure 2**). Isocitrate dehydrogenases are cytoplasmic (IDH1) and mitochondrial (IDH2) enzymes cathalyse the reduction of a-ketoglutarate (a-KG) to citrate in krebs cycle in a NADPH-dependent way. NADPH is important for the reduction of glutathione, which in the reduced state is a major antioxidant and protects the cell against reactive-oxygen species (ROS) and other free radicals. IDH mutations have a loss of function effect, producing the accumulation of the oncometabolite R2-hydroxyglutarate (2-HG) which competitively inhibits multiple α-ketoglutarate dependent dioxygenases such as lysine (K)-specific demethylase (KDM) and ten eleven translocation methylcytosine dioxygenase 2 (TET2), causing widespread epigenetic changes with global dysregulation of gene expression and abnormal differentiation and proliferation of leukemic cells (**Figure 3**) [7]. Furthermore 2-HG activates the EglN family of prolyl 4-hydroxylases (EglN), with consequent ubiquitination and degradation of HIF1a, impairing p53 dependent apoptosis. IDH1

**290**

**Figure 2.**

*p53 mitochondrial pathway and mechanisms of resistance to Venetoclax (light yellow labels).*

*Mechanism of leukemogenesis of IDH mutations (green label) and mechanisms of resistance to IDHi (light yellow label).*

mutations also result in a lack of crucial metabolites including a decrease in the NADPH pool and inhibition of krebs cycle with metabolic changes conferring chemotherapy resistance of leukemic cell. At last 2-HG determines a leukemic status highly BCL-2 dependent, preventing the hypoxia mediated apoptosis, determined by cytochrome c oxidase inhibiton.

Recent studies utilizing NGS and single-cell technologies have also illustrated the complex and polyclonal nature of resistance to targeted therapeutics including FLT3, BCL2 and IDH inhibitors (FLT3i, BCL2i, IDHi) [8, 9]. Here we report the results of the principle studies aiming to analyze mechanism of primary and secondary leukemic resistance to new approved target therapies.

#### **2. New target therapies in AML**

#### **2.1 FLT3 inhibitors (FLT3i)**

FLT3 is a Tirosin Kinase receptor expressed by hematopoietic progenitors and mutated in 25-30% AML. The mutations involve two different domains: the iuxtamembrane domain (FLT3 ITD) in 20-25% AML and the tirosin kinase domain (TKD) in 5-10% AML, expecially at codon D835. They both determine the constitutive activation of the FLT3 receptor tyrosine kinase, inducing cellular proliferation and survival and inhibiting differentiation, through the activation of PI3K/AKT/ mTOR pathway, with a critical role in leukemogenesis [10] (**Figure 1**).

#### *Acute Leukemias*

Target drugs inhibiting FLT3 receptor showed different potency of inhibition, activity on FLT3-ITD versus TKD mutations, and on non-FLT3 targets (i.e., kinome specificity), with variable off-target toxicities [11].

Type I FLT3i (Lestaurtinib, Midostaurin, Gilteritinib, Crenolanib) are active against both FLT3-ITD and TKD mutations because they interact with the gatekeeper domain near to the activation loop or with the ATP binding site, expleting their activity on both active dimeric and inactive monomeric tirosin kinase receptor. Type II FLT3i (Quizartinib and Sorafenib) bind to the hydrophobic region adjacent to the ATP binding site only when the receptor is in an inactive form and are therefore ineffective in the forms with the FLT3TKD mutations where the receptor is always in the dimeric active form.

### *2.1.1 Midostaurin*

Midostaurin, a type I FLT3i, also targets c-KIT, PKC, PDGFR, and VEGFR [12] and is FDA, EMA and AIFA approved for the first line treatment of FLT3 mutated (FLT3-mut) AML in association with 7 + 3 in induction and high dose Cytarabine in consolidation, on the basis of the results of the multinational, randomized phase III trial RATIFY (CALBG 10603) [13]. Midostaurin or placebo were given during induction and consolidation, and could be given for up to one year as postconsolidation maintenance, allogeneic transplant was admitted after the stop of the experimental treatment. Midostaurin was associated with a significant improvement in OS (4-year OS rate: 51.4% versus 44.3%; median OS: 74.7 months versus 25.6 months, HR = 0.78; P = 0.009) regardless of the type of *FLT3* mutation (e.g., ITD or D835 TKD) or ITD allele burden, (<0.7/≥0.7).

#### *2.1.2 Quizartinib*

Quizartinib is a type II FLT3i, but also a potent inhibitor of c-KIT, PDGFR, and RET achieving 45-50% marrow remission rates as single-agent in relapsed/ refractory (R/R) *FLT3*-mut AML with an OS advantage over investigator choice salvage chemotherapy in the Quantum R-trial, a phase III randomized study of 367 patients with relapsed or refractory *FLT3-*ITD mutated AML (CRc rate 48% vs. 27%; median OS 6.2 months vs. 4.7 months, P = 0.0177) [14]. Neverthless Quizartinib failed FDA approval for this indication, due in part to concerns over treatment equipoise and robustness of OS improvement, while obtained approval in Japan in June 2019 and is being considered for approval in other countries.

#### *2.1.3 Gilteritinib*

Gilteritinib is another potent second-generation type I inhibitor with activity against AXL, a receptor tyrosine kinase that may play a role in mediating resistance to earlier generation FLT3 inhibitors [15]. Gilteritinib was found to be well tolerated as single-agent in a randomized phase III study enrolling R/R FLT3-mut AML, with marrow remission rates of 54% superior to the 22% CRc rate observed after investigator choice salvage chemotherapy (both high- and low-dose chemotherapy), with also a longer median OS (9.3 months vs. 5.6 months, HR = 0.79; P = 0.007) [16]. More patients (26% vs. 15%) were able to proceed to HSCT with gilteritinib compared with salvage chemotherapy. These results led to Gilteritinib FDA approval for the treatment of R/R FLT3-mut AML (both ITD and TKD) in November 2018.

**293**

*Mechanisms of Resistence of New Target Drugs in Acute Myeloid Leukemia*

Some concerns on these last two trials have been recently raised by a french retrospective analysis of 160 patients with R/R (114 relapsed and 46 refractory) FLT3-mut AML after a first-line TKI-free treatment, 92 of whom fulfilling the main criteria of the QUANTUM-R study, with CR1 durations <6 months, who received an intensive salvage regimen in 48.9% of cases achieving a 52.8% CRc rate and a bridge to transplant rate of 39.6%, superior to 27% of CR and 11% of bridge to transplant rates observed in the same setting in QUANTUM-R. The Median OS of 7 months observed in the French study was also superior to the Quantum-R OS of 4.7 months. The authors argue that the possible bias, caused by the inclusion in the control arm of patients receiving low-intensity regimens, such as low-dose cytarabine or hypomethylating agents, might compromise the results of similar

Neverthless hematopoietic stem cell transplant (HSCT) is still necessary and recommended for the cure of the disease, since retrospective studies [18, 19] showed that HSCT improves RFS and OS and reduces incidence of relapse. The favorable predictive role of FLT3 allelic ratio in NPM1 mutated AML is still controversial due to lack of standardization of techniques and thresholds of this factor [20, 21]. Novel FLT3i might increase outcomes in this setting, but researchers have already identified multiple mechanisms of resistance as hereby reported [11].

• The acquisition of secondary mutations of single amino acids of the activation loop of the FLT3 receptor (D835, I836, D839, Y842) or of the gate-keeper residue (F691) called 'TKD' mutations are reported in 22% of FLT3 AML [22] and are responsible for the resistance specially to type II FLT3i (Quizartinib

• The activity of fibroblast growth factor 2 (FGF2) and CXCL12/CXCR4 pathways in FLT3 mutated leukemic cells can induce their chemoresistance. The increase in FGF2 is an autocrine response mechanism of stromal cells to all phenomena of hematopoietic stress, including that induced by Quizartinib. Paracrine production of FL (FLT3 ligand) by stromal cells also inhibits the action of FLT3 inhibitors with competitive mechanism, but removal of FL from stromal and leukemic cell cultures does not stop the chemoresistance process due to activation of RAS-MAPK mediated by the FGF2-FGFR1 interaction. The increase in FGF2 secreted by stromal cells has been reported to precede the relapse of mutated FLT3ITD mut AML treated with Quizartinib, through activation of the RAS–MEK/MAPK signal [25]. The combination of FGFR and FLT3 inhibitors is being studied, the rationale is represented by the inhibition of the autocrine and paracrine stimulus favoring the survival of the stromal

• Furthermore FLT3ITD mutated leukemic cells express CXCR4 and are CXCL12 dependent for growth and survival, which makes them resistant to the action of chemotherapy [26]. Activation of Nutlin-3a reduces mRNA levels and CXCL12 secretion through activation of p53 and consequent down-regulation of HIF-1 alpha. Nutlin-3a also binds MDM2 in the p53 binding domain,

inhibiting its interaction with p53 which, remaining free, recovers its function.

and Sorafenib) ineffective in targeting TKD mutations [23, 24].

*2.1.4 Considerations on phase III trials in R/R FLT3-mut AML*

*DOI: http://dx.doi.org/10.5772/intechopen.94978*

phase 3 trials [17].

*2.1.5 Mechanisms of resistance to FLT3i*

and leukemic cells, respectively.

*Mechanisms of Resistence of New Target Drugs in Acute Myeloid Leukemia DOI: http://dx.doi.org/10.5772/intechopen.94978*

### *2.1.4 Considerations on phase III trials in R/R FLT3-mut AML*

Some concerns on these last two trials have been recently raised by a french retrospective analysis of 160 patients with R/R (114 relapsed and 46 refractory) FLT3-mut AML after a first-line TKI-free treatment, 92 of whom fulfilling the main criteria of the QUANTUM-R study, with CR1 durations <6 months, who received an intensive salvage regimen in 48.9% of cases achieving a 52.8% CRc rate and a bridge to transplant rate of 39.6%, superior to 27% of CR and 11% of bridge to transplant rates observed in the same setting in QUANTUM-R. The Median OS of 7 months observed in the French study was also superior to the Quantum-R OS of 4.7 months. The authors argue that the possible bias, caused by the inclusion in the control arm of patients receiving low-intensity regimens, such as low-dose cytarabine or hypomethylating agents, might compromise the results of similar phase 3 trials [17].
